Photoswitchable HDAC inhibitors

ABSTRACT

This invention relates to photoswitchable inhibitors of histone deacetylases and methods of using the same.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.15/968,634, filed May 1, 2018, which is a continuation of U.S. patentapplication Ser. No. 14/775,340, filed Sep. 11, 2015, now U.S. Pat. No.9,981,038, which is a U.S. National Phase Application under 35 U.S.C. §371 of International Patent Application No. PCT/US2014/026069, filed onMar. 13, 2014, which claims the benefit of U.S. Provisional ApplicationSer. No. 61/780,373, filed on Mar. 13, 2013, all of which areincorporated herein by reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos.R01DA028301, P50A086355, and T32-CA079443 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

TECHNICAL FIELD

This invention relates to photoswitchable inhibitors of histonedeacetylases and methods of using the same.

BACKGROUND

Small molecule modulators of biomacromolecule function, in particular,receptors and enzymes, are widely used both as drugs to treat humandiseases and as tool compounds to study biological processes.Importantly, small molecules enable scientific approaches that offerhigh spatial and temporal resolution, which can rarely be achieved withtraditional molecular biological methods. However, when used in thecontext of larger biological settings such as organs or whole organisms,the spatiotemporal resolution of small molecules is limited and may beinsufficient, resulting in side effects (when used as a drug) andindefinite experimental outcomes (when used as a tool). Thus, approachesthat will provide an additional level of control with respect to timeand space are highly desirable.

SUMMARY

Provided herein are inhibitors of histone deacetylases (HDACs) havingphoto-switchable modulators of protein function with short thermalrelaxation kinetics. In some embodiments, the compounds provided hereinare characterized as having a ground state, i.e. thermally relaxed,trans-isomer which is less active (e.g., inactive) compared to theexcited state cis-isomer.

Provided herein is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof,wherein:

-   X and Y are independently a substituted or unsubstituted aryl or    heteroaryl ring, wherein at least one of the rings is substituted    with one or more HDAC targeting elements.

In some embodiments, Y is substituted with one or more HDAC targetingelements and X is substituted with one or more fluorescent moieties.

In some embodiments, a compound of Formula (I) is a compound of Formula(II):

or a pharmaceutically acceptable salt thereof,wherein:

-   each R¹ is independently selected from the group consisting of:    hydrogen, C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkynyl, halo, C₁₋₆ haloalkyl,    CN, NO₂, OR⁴, SR⁴, C(O)R⁴, C(O)NR⁴R⁵, C(O)OR⁴, OC(O)R⁴, OC(O)NR⁴R⁵,    C(═NR⁴)NR⁵R⁶, NR⁴C(═NR⁵)NR⁶R⁷, NR⁴R⁵, NR⁴C(O)R⁵, NR⁴C(O)OR⁵,    NR⁴C(O)NR⁵R⁶, NR⁴S(O)R⁵, NR⁴S(O)₂R⁵, NR⁴S(O)₂NR⁵R⁶, S(O)R⁴,    S(O)NR⁴R⁵, S(O)₂R⁴, S(O)₂NR⁴R⁵, C₁₋₆alkoxyalkyl, carbocyclyl,    C₁₋₆carbocyclylalkyl, heterocyclyl, C₁₋₆heterocyclylalkyl, aryl,    C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl;-   R² is an HDAC targeting element;-   R³ is independently selected from the group consisting of:    C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, halo, C₁₋₆ haloalkyl, CN, NO₂,    OR⁴, SR⁴, C(O)R⁴, C(O)NR⁴R⁵, C(O)OR⁴, OC(O)R⁴, OC(O)NR⁴R⁵,    C(═NR⁴)NR⁵R⁶, NR⁴C(═NR⁵)NR⁶R⁷, NR⁴R⁵, NR⁴C(O)R⁵, NR⁴C(O)OR⁵,    NR⁴C(O)NR⁵R⁶, NR⁴S(O)R⁵, NR⁴S(O)₂R⁵, NR⁴S(O)₂NR⁵R⁶, S(O)R⁴,    S(O)NR⁴R⁵, S(O)₂R⁴, S(O)₂NR⁴R⁵, C₁₋₆alkoxyalkyl, carbocyclyl,    C₁₋₆carbocyclylalkyl, heterocyclyl, C₁₋₆heterocyclylalkyl, aryl,    C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl;-   each R⁴, R⁵, and R⁶ are independently selected from H and C₁₋₆alkyl;-   m is an integer from 0 to 4; and-   n is an integer from 1 to 5.

In some embodiments, R¹ is an electron withdrawing group. For example,R¹ can be independently selected from the group consisting of: NR⁵R⁶,OR⁵, SR⁵, C₁₋₆ alkyl, CH═N—NR⁵R⁶, CH═C(NR⁵R⁶)₂, NR⁵COR⁶, NR⁵C(O)NR⁶R⁷,aryl, and heteroaryl; wherein each R⁵, R⁶, and R⁷ is independentlyselected from H and C₁₋₆ alkyl. In some embodiments, n is 1 and R¹ is inthe para position on the ring. In some embodiments, R¹ is NR⁵R⁶.

In some embodiments, m is 0 and R² is in the ortho position on the ring.In some embodiments, m is 1 and R² is in the ortho position and the R³is in the meta position across the ring from the first R².

In some embodiments, R¹ and/or R³ can be independently selected fromcarbocyclyl, C₁₋₆carbocyclylalkyl, heterocyclyl, C₁₋₆heterocyclylalkyl,aryl, C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl. For example, R¹and/or R³ can be independently selected from a substituted orunsubstituted tetrazine, a substituted or unsubstitutedtrans-cyclooctene, and a substituted or unsubstituted cyclopropene.

In some embodiments, the HDAC targeting element is selected from thegroup consisting of: a substituted or unsubstituted aminobenzamide, asubstituted or unsubstituted hydroxybenzamide, and hydroxamic acids. Forexample, the HDAC targeting element is selected from the groupconsisting of: CI-994, Entinostat (MS-275), HDAC1/2 selective CI-994analog, Mocetinostat (MGCD0103), and analogs thereof.

Also provided herein is a compound of Formula (IV):

or a pharmaceutically acceptable salt thereof,wherein:

-   each R¹ is independently an electron donating substituent;-   each R² is independently selected from the group consisting of:    halogen, NR³R⁴, OR³, aryl, and heteroaryl;-   each R³ and R⁴ is independently selected from the group consisting    of: H, C₁₋₆ alkyl, and a nitrogen protecting group;-   m is an integer from 1 to 5; and-   n is an integer from 1 to 5.

This disclosure also provides a compound of Formula (V):

or a pharmaceutically acceptable salt thereof,wherein:

-   R¹ is an electron donating substituent;-   R² is selected from the group consisting of: NR¹⁰R¹¹ and OR¹⁰;-   each R³ is independently selected from the group consisting of:    hydrogen, C₁₋₉alkyl, C₂₋₉alkenyl, C₂₋₉alkynyl, halo, C₁₋₉ haloalkyl,    CN, NO₂, OR⁷, SR⁷, C(O)R⁷, C(O)NR⁷R⁸, C(O)OR⁷, OC(O)R⁷, OC(O)NR⁷R⁸,    C(═NR⁷)NR⁸R⁹, NR⁷C(═NR⁸)NR⁹R⁹, NR⁷R⁸, NR⁷C(O)R⁸, NR⁷C(O)OR⁸,    NR⁷C(O)NR⁸R⁹, NR⁷S(O)R⁸, NR⁷S(O)₂R⁸, NR⁷S(O)₂NR⁸R⁹, S(O)R⁷,    S(O)NR⁷R⁸, S(O)₂R⁷, S(O)₂NR⁷R⁸, C₁₋₉alkoxyalkyl, carbocyclyl,    C₁₋₉carbocyclylalkyl, heterocyclyl, C₁₋₉heterocyclylalkyl, aryl,    C₁₋₉aralkyl, heteroaryl, and C₁₋₉heteroaralkyl;-   each R⁴ is independently selected from the group consisting of: H,    halogen, aryl, and heteroaryl;-   each R⁷, R⁸, and R⁹ is independently selected from the group    consisting of: H, C₁₋₆ alkyl;-   R¹⁰ and R¹¹ are independently selected from the group consisting of:    H, C₁₋₆ alkyl, and a nitrogen protecting group; and-   p is an integer from 0 to 4.

Non-limiting examples of a compound of Formula (I) include:

or a pharmaceutically acceptable salt form thereof.

The compounds and compositions provided herein are also useful fortreating proliferative disorders (e.g., cancer, including skin cancer,and retinal disorders). Accordingly, a method is provided hereincomprising administering a therapeutically effective amount of acompound of Formula (I) to the patient. For example, the method caninclude administering a therapeutically effective amount of a compoundof Formula (I) to the patient; and exposing the patient to lightsuitable to convert the compound of Formula (I) to its cis confirmation.In some embodiments, the light exposure occurs at a location containingthe cancer (e.g., the patient's skin).

In some embodiments, a compound provided herein can be used to inhibitan HDAC in a cell. The method can include contacting the cell with aneffective amount of a compound of Formula (I). In some embodiments, themethod can further include exposing the cell to light suitable toconvert the compound of Formula (I) to its cis confirmation.

By virtue of their design, the compounds, compositions, and methodsprovided herein possess certain advantages and benefits. The compoundsdescribed herein can provide high spatiotemporal resolution in thetreatment of various disease states (e.g., skin cancer and retinaldisorders). Additionally, the compounds described herein can targettreatment to specific areas of the body and therefore exhibit lowerundesirable side effects.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 illustrates differences between the trans and cis isomers ofazobenzene.

FIG. 2 illustrates the absorbance overlap between the trans and cisisomers of azobenzene.

FIG. 3 illustrates differences between the trans and cis isomers ofunsubstituted and substituted azobenzenes.

FIG. 4 shows a 12×8 LED array developed to match standardized microtiterplate layouts.

FIG. 5 illustrates the absorbance spectra of selected compoundsdescribed herein (BG14, BG18, BG12, and BG19).

FIG. 6 shows the inhibitory activity of selected compounds against HDAC1with and without exposure (including 1 hr pre-exposure) to 470 nm light.

FIG. 7 shows the inhibitory activity of selected compounds against HDAC2with and without exposure (including 1 hr pre-exposure) to 470 nm light.

FIG. 8 shows the inhibitory activity of selected compounds against HDAC3with and without exposure (including 1 hr pre-exposure) to 470 nm light.

FIG. 9 compares the activity change of BG14 and the reference HDACinhibitors MS-275 and SAHA as a function of light exposure time.

FIG. 10 illustrates the long residence time of the compounds onceengaged with an HDAC enzyme as shown for BG47 and BG48.

FIG. 11 shows the activity of the compounds in live cells usingacetylation of histone H3 Lysine 9 as a biomarker for HDAC inhibition(immunofluorescent staining).

FIG. 12 shows the activity of the compounds in live cells usingacetylation of histone H3 Lysine 9 as a biomarker for HDAC inhibition.(western blotting)

FIGS. 13A-13F provide (FIG. 13A) measurement of thermal relaxationhalf-life, (FIG. 13B) the LED array, (FIG. 13C) light intensity andconcentration dependent inhibition of HDAC3 by BG14, and (FIGS. 13D-13F)light-dependent activity profiles of the tested compounds againstHDAC1-3.

FIGS. 14A-14E provide (FIGS. 14A-14C) Western Blot analysis foracetylH3K9, (FIG. 14D) immunofluorescence staining, and (FIG. 14E)Quantification of the cell staining shown in FIG. 14D.

FIGS. 15A-15G provide a (FIG. 15A) representative example forlight-dependent increase of gene transcription by BG14, (FIG. 15B)representative example for light-dependent decrease of genetranscription by BG14, (FIG. 15C) gene expression analysis demonstratesthat transcriptional changes are light dependent. (FIG. 15D)Gene-Set-Enrichment-Analysis shows BG14 is highly similar to a standardHDAC inhibitor (CI-994) in the presence of light. (FIG. 15E)Venn-Diagram of genes regulated by CI-994 and BG14 with and withoutlight. (FIG. 15F) Cellular networks regulated by BG14 in the presence oflight. (FIG. 15G) BG14 regulates cell cycle network in the presence oflight.

DETAILED DESCRIPTION Definitions

For the terms “for example” and “such as,” and grammatical equivalencesthereof, the phrase “and without limitation” is understood to followunless explicitly stated otherwise. All measurements reported herein areunderstood to be modified by the term “about”, whether or not the termis explicitly used, unless explicitly stated otherwise. As used herein,the singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

As used herein, “alkyl” means a branched, or straight chain chemicalgroup containing only carbon and hydrogen, such as methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl,n-pentyl, iso-pentyl, sec-pentyl and neo-pentyl. Alkyl groups can eitherbe unsubstituted or substituted with one or more substituents, e.g.,halo, alkoxyl, acyloxyl, amino, amido, cyano, nitro, hydroxyl, thiol,carboxyl, carbonyl, benzyloxy, aryl, heteroaryl, and with one or morefluorescent moieties. Alkyl groups can be saturated or unsaturated(e.g., containing —C═C— or —C≡C— subunits), at one or several positions.Typically, alkyl groups will comprise 1 to 6 carbon atoms, for example,1 to 4 or 1 to 2 carbon atoms.

As used herein, “aryl” means an aromatic radical having a single-ring(e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl)with only carbon atoms present in the ring backbone. Aryl groups caneither be unsubstituted or substituted with one or more substituents,e.g., amino, cyano, hydroxyl, lower alkyl, haloalkyl, alkoxyl, nitro,halo, thiol, and other substituents. In some embodiments, an aryl isphenyl.

As used herein, the term “heteroaryl” means an aromatic radical havingone or more heteroatom(s) (e.g., N, O, or S) in the ring backbone andmay include a single ring (e.g., pyridine) or multiple condensed rings(e.g., quinoline). Heteroaryl groups can either be unsubstituted orsubstituted with one or more substituents, e.g., amino, cyano, hydroxyl,alkyl, haloalkyl, alkoxyl, nitro, halo, thiol, and other substituents(e.g., one or more fluorescent moieties). Examples of heteroaryl includethienyl, pyridinyl, furyl, oxazolyl, oxadiazolyl, pyrrolyl, imidazolyl,triazolyl, thiodiazolyl, pyrazolyl, isoxazolyl, thiadiazolyl, pyranyl,pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thiazolyl benzothienyl,benzoxadiazolyl, benzofuranyl, benzimidazolyl, benzotriazolyl,cinnolinyl, indazolyl, indolyl, isoquinolinyl, isothiazolyl,naphthyridinyl, purinyl, thienopyridinyl, pyrido[2,3-d]pyrimidinyl,pyrrolo[2,3-b]pyridinyl, quinazolinyl, quinolinyl,thieno[2,3-c]pyridinyl, pyrazolo[3,4-b]pyridinyl,pyrazolo[3,4-c]pyridinyl, pyrazolo[4,3-c]pyridine,pyrazolo[4,3-b]pyridinyl, tetrazolyl.

As used herein, “halo”, “halide” or “halogen” is a chloro, bromo, fluoroor iodo atom radical.

As used herein, “haloalkyl” means a hydrocarbon substituent, which islinear or branched or cyclic alkyl, alkenyl or alkynyl substituted withchloro, bromo, fluoro, or iodo atom(s).

The skilled artisan will recognize that some structures described hereinmay be resonance forms or tautomers of compounds that may be fairlyrepresented by other chemical structures, even when kinetically, theartisan recognizes that such structures are only a very small portion ofa sample of such compound(s). Such compounds are clearly contemplatedwithin the scope of this invention, though such resonance forms ortautomers are not represented herein.

The compounds provided herein may encompass various stereochemicalforms. The compounds also encompasses diastereomers as well as opticalisomers, e.g. mixtures of enantiomers including racemic mixtures, aswell as individual enantiomers and diastereomers, which arise as aconsequence of structural asymmetry in certain compounds. Separation ofthe individual isomers or selective synthesis of the individual isomersis accomplished by application of various methods which are well knownto practitioners in the art. Unless otherwise indicated, when adisclosed compound is named or depicted by a structure withoutspecifying the stereochemistry and has one or more chiral centers, it isunderstood to represent all possible stereoisomers of the compound.

The term “administration” or “administering” refers to a method ofgiving a dosage of a compound or pharmaceutical composition to avertebrate or invertebrate, including a mammal, a bird, a fish, or anamphibian, where the method is, e.g., orally, subcutaneously,intravenously, topically, transdermally, intraocularly,subconjuctivally, via anterior eye chamber injection, andintravitreally, inhalation. The method of administration can varydepending on various factors, for example, the components of thepharmaceutical composition, the site of the disease, the diseaseinvolved, and the severity of the disease.

The term “mammal” is used in its usual biological sense. Thus, itspecifically includes humans, cattle, horses, dogs, and cats, but alsoincludes many other species.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, co-solvents,complexing agents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the likewhich are not biologically or otherwise undesirable. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions. In addition, various adjuvantssuch as are commonly used in the art may be included. These and othersuch compounds are described in the literature, e.g., in the MerckIndex, Merck & Company, Rahway, N.J. Considerations for the inclusion ofvarious components in pharmaceutical compositions are described, e.g.,in Gilman et al. (Eds.) (2010); Goodman and Gilman's: ThePharmacological Basis of Therapeutics. 12th Ed., The McGraw-HillCompanies.

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of the compounds of thepreferred embodiments and, which are not biologically or otherwiseundesirable. In many cases, the compounds of the preferred embodimentsare capable of forming acid and/or base salts by virtue of the presenceof amino and/or carboxyl groups or groups similar thereto.Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids. Inorganic acids from which salts canbe derived include, for example, hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acidsfrom which salts can be derived include, for example, acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and thelike. Pharmaceutically acceptable base addition salts can be formed withinorganic and organic bases. Inorganic bases from which salts can bederived include, for example, sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum, and thelike; particularly preferred are the ammonium, potassium, sodium,calcium and magnesium salts. Organic bases from which salts can bederived include, for example, primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines, basic ion exchange resins, and the like, specificallysuch as isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, and ethanolamine. Many such salts are known in the art,as described in WO 87/05297.

“Patient” as used herein, means a human or a non-human mammal, forexample, a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, anon-human primate or a bird, for example, a chicken, as well as anyother vertebrate or invertebrate.

A “therapeutically effective amount” or “pharmaceutically effectiveamount” is one the amount of a compound provided herein which issufficient to achieve the desired effect and may vary according to thenature and severity of the disease condition, and the potency of thecompound. “Therapeutically effective amount” is also intended to includeone or more of the compounds in combination with one or more otheragents that are effective to inhibit HDAC related diseases and/orconditions. The combination of compounds is preferably a synergisticcombination. Synergy, as described, for example, by Chou and Talalay,Advances in Enzyme Regulation (1984), 22, 27-55, occurs when the effectof the compounds when administered in combination is greater than theadditive effect of the compounds when administered alone as a singleagent. In general, a synergistic effect is most clearly demonstrated atsub-optimal concentrations of the compounds. It will be appreciated thatdifferent concentrations may be employed for prophylaxis than fortreatment of an active disease. This amount can further depend upon thepatient's height, weight, sex, age and medical history.

A therapeutic effect relieves, to some extent, one or more of thesymptoms of the disease, and includes curing a disease. “Curing” meansthat the symptoms of active disease are eliminated. However, certainlong-term or permanent effects of the disease may exist even after acure is obtained (such as extensive tissue damage).

“Treat,” “treatment,” or “treating,” as used herein refers toadministering a compound provided herein or a pharmaceutical compositioncomprising the same for therapeutic purposes. The term “therapeutictreatment” refers to administering treatment to a patient alreadysuffering from a disease thus causing a therapeutically beneficialeffect, such as ameliorating existing symptoms, ameliorating theunderlying metabolic causes of symptoms, postponing or preventing thefurther development of a disorder and/or reducing the severity ofsymptoms that will or are expected to develop.

The term “contacting” means bringing at least two moieties together,whether in an in vitro system or an in vivo system.

Compounds

Light is a reagent that can be applied with unparalleled precision, andthere have been various reports using light as an activator of smallmolecules (e.g. caged inhibitors) and proteins (e.g. rhodopsin) (Brieke,C. et al., Angew. Chem. Int. Ed. 2012, 2-34). More recently smallmolecules that feature unique chemical motifs, which can be switchedreversibly between two distinct structural conformations (i.e. shapes orgeometries) upon exposure to light of a specific wavelength, haveattracted attention in protein engineering and small molecule inhibitordesign (Brieke, C. et al., Angew. Chem. Int. Ed. 2012, 2-34; andBeharry, A. A. and Woolley, G. A., Chem. Soc. Rev. 2011, 40, 4422).Provided that the two structural confirmations exhibit distinctbiological activity states (ideally active and inactive) and suitablephotophysical properties, light can be used to modulate the activity ofthe respective compounds in physiological settings with high resolutionenabling experiments probing biological events with sub-cellularprecision and on millisecond timescales.

Many challenges are present in the development of such molecules. Forexample, the two molecular states (ground and excited) should exhibit alarge difference in potency, ideally several orders of magnitude. Inaddition, switching between the two states should be efficient andswitching to the low activity state (switch off) should be close tocomplete.

One well-studied photo-switchable compound class is based on azobenzene(see FIG. 1 ). The thermodynamic ground state of azobenzenes favors atrans geometry. Upon exposure to light of an appropriate wavelength,usually ultraviolet light, azobenzenes switch to an energetically highercis geometry, a process referred to as photoisomerization (Beharry, A.A. and Woolley, G. A., Chem. Soc. Rev. 2011, 40, 4422). This process canbe reverted by exposure to light of appropriate (generally longer)wavelength (Brieke, C. et al., Angew. Chem. Int. Ed 2012, 2-34; andBeharry, A. A. and Woolley, G. A., Chem. Soc. Rev. 2011, 40, 4422).Switching between cis- and trans-geometry has a profound impact on theoverall shape, orientation of substituents and electronic properties ofthe molecule (see FIG. 1 ). The cis and trans isomers of biologicallyactive azobenzenes that exploit any of these molecular characteristicsfor target binding are consequently expected to have significantlydifferent potencies Beharry, A. A. and Woolley, G. A., Chem. Soc. Rev.2011, 40, 4422; and Bandara, H. M. D.; Burdette, S. C., Chem. Soc. Rev.2012, 41, 1809).

Unfortunately, photo-induced switching of azobenzenes between cis andtrans geometry is not quantitative. The maximum ratio between the twogeometries correlates with the inverse ratio of absorbance coefficientsof both isomers at the wavelength that is used for switching (see FIG. 2). Because the absorbance spectra of the cis and trans isomers overlapit is generally only possible to change the cis/trans ratio within oneorder of magnitude (e.g., between 9:1 and 1:9, respectively) andtherefore the ability to modulate the activity of such a compound bylight is confined within this range. In contrast to photo-inducedrelaxation, thermal relaxation is quantitative. Generally, thecis-azobenzene is thermodynamically less favored and will thermallyrelax back to the more stable trans isomer. The rate constant of thisprocess, amongst other factors such as solvent polarity and pH, isdependent on the electronic properties of the substituents attached tothe azobenzene core and can range from microseconds to months (see FIG.3 ) (Brieke, C. et al., Angew. Chem. Int. Ed 2012, 2-34; and Beharry, A.A. and Woolley, G. A., Chem. Soc. Rev. 2011, 40, 4422).

Much of the development to date has focused on the synthesis ofdiazobenzenes with medium to long thermal relaxation half-lives (secondsor longer), while compounds with short (milliseconds) and very short(sub-milliseconds) thermal relaxation half-lives are generallyconsidered less attractive (Brieke, C. et al., Angew. Chem. Int. Ed.2012, 2-34; and Beharry, A. A. and Woolley, G. A., Chem. Soc. Rev. 2011,40, 4422). One reason for this is that maintaining a significantpopulation of the cis-isomer of a photoswitchable compound with fastthermal relaxation requires very high light intensity (resulting inoverheating of the biological specimen) and the concentration ofcis-isomer could very rapidly decline immediately after termination oflight exposure. This feature, however, can be an advantage whenexploited properly. The rapid isomerization back to the ground (inactivestate) limits the off target effects that can arise from activepharmacological agents circulating in the body and interacting withother biomacromolecules as is the case for bare therapeutics, prodrugs,and caged compounds.

Generally azobenzenes with short thermal relaxation half-lives are socalled push-pull systems (type III). These are functionalizedazobenzenes that carry an electron-donating substituent on one arylgroup and an electron-withdrawing substituent on the other. Azobenzenes,push-push or pull-pull substitution patterns (type II) have longerthermal relaxation kinetics, while those azobenzenes with none orelectroneutral substituents (type I) are most stable.

Azobenzenes with push-pull substitution patterns have a number ofsuperior properties compared to the other classes of azobenzenes. Theygenerally absorb longer wavelength light compared to the other classes,which tend to require tissue damaging violet to UV-light. Furthermore,type III azobenzenes absorb light more efficiently (i.e. they have thehighest absorbance coefficient). Lastly, the disruption of the push-pullsystem, which is the consequence of adopting the cis-confirmation,causes the strongest relative change of the electronic property of thearomatic system and substituents (see FIG. 3 ). Therefore, if electroniceffects are important for targeting and engagement of the compound, alarge difference in affinity between the cis and trans isomer isexpected. The affinity of a small molecule ligand is defined by theequilibrium dissociation constant K_(D) that represents the fraction ofthe dissociation rate (k_(off)) over the association rate (k_(on)).Small molecule inhibitors generally exhibit relatively fast on- andoff-rates. As a result the equilibrium of bound and unbound inhibitor isreached within seconds. There are many small molecule ligands derivedfrom a wide variety of inhibitor classes, however, that arecharacterized by slow binding kinetics. The dissociation half-lives ofsuch ligand-protein complexes can range from minutes to hours or evendays. This critical feature is frequently not appreciated. Inparticular, in therapeutic settings long dissociative half-lives can bevery desirable to help ensure long-term target inhibition for drugshaving a short systemic exposure (Swinney, D. PART VI: Topics inBiology-18 Molecular Mechanism of Action (MMoA) in Drug Discovery;Annual Reports in Medicinal Chemistry, 2011; Swinney, D. C.Pharmaceutical Medicine 2008, 22, 23-34; and Copeland, R. A.; Pompliano,D. L.; Meek, T. D. Nat Rev Drug Discov 2006, 5, 730-739).

As provided herein, the terms “slow” and “fast” used to describe thethermal relaxation kinetics of azobenzenes and the binding kinetics of asmall molecule ligand to its biological target, respectively, arerelative descriptors for each process. The two processes occur ondifferent time scales and “very fast” thermal relaxation is a “veryslow” process (t_(1/2)=millisecond time scale) with respect to ligandbinding kinetics of a ligand with “slow” on rates (k_(on)=10⁸ M⁻¹ s⁻¹).

Comparing the events on an absolute time scale it is more likely for anactivated cis-azobenzene ligand with fast relaxation kinetics to bind toits target (provided affinity) than to thermally relax to the inactivetrans-isomer. The target proteins are acting similar to a sponge thatscavenges the cis-isomers until the binding capacity is reached. Oncebound, the cis-isomer is stabilized by ligand-target interaction andpossibly sterically locked (similar to a jack-in-the-box), preventingrelaxation to the inactive isomer while bound to the target protein.Thus, ligands with long target residence time will cause prolongedtarget inhibition following short term light exposure independent fromthe thermal relaxation kinetics of the respective cis-azobenzene. Oneadvantage of fast relaxing azobenzenes in addition to favorablephotophysical properties (benign wavelength, high absorbancecoefficient) is the immediate self-deactivation of the respectiveligands, which are not engaged with a target protein, once exposure tolight is ceased or if the activated molecule diffuses outside thelight-exposed area. Activity of corresponding ligands with longerthermal relaxation rates would fade off slowly, providing less controland potentially causing remote target effects.

Provided herein are a set of compounds that are structurally related toa specific class of histone deacetylases (HDAC) inhibitors, whichfeature a common amino-benzamide or hydroxy-benzamide pharmacophor. ThisHDAC inhibitor class includes, for example, CI-994 and MS-275.

Benzamides generally show remarkable preference for HDACs1-3 over otherHDAC isoforms. Further selectivity and increased potency for HDAC1/2over HDAC3 can be attained by introduction of aryl substituents in the4′-position (Moradei, O. M. et al., J. Med. Chem. 2007, 50, 5543-5546;and Witter, D. J. et al., Bioorg. Med. Chem. Lett. 2008, 18, 726-731).These modification exploit isoform specific features within an internalcavity that is adjacent to the catalytic zinc and proposed to serve as arelease pathway for acetate following enzymatic hydrolysis (Wang, D.-F.et al., J. Med. Chem. 2004, 47, 3409-3417; Tessier, P. et al., Bioorg.Med. Chem. Lett. 2009, 19, 5684-5688; and Bressi, J. C. et al., Bioorg.Med. Chem. Lett. 2010, 20, 3142-3145). Phenyl-, 2-thienyl and2-furyl-substituents have been identified by both groups as attractivepharmacophores. In contrast to other HDAC inhibitors, benzamides arecharacterized by long target residence time.

As shown below, the compounds provided herein are designed as hybridsbetween a generic benzamide HDAC inhibitor and the azobenzene motif,sharing a common electron-withdrawing (pull system) on one end.

This design is advantageous for a number of reasons. The amino-benzamideis acting as a chelator that binds the catalytic zinc in the activesite. The binding affinity of the chelator is strongly dependent on theelectronic properties of the benzamide. Electron-rich benzamides aregenerally low affinity ligands, while electron neutral orelectron-withdrawing ligands result in increased affinity. It isexpected that the hybrid molecule is electron rich as a result of theconjugation of both aromatic systems. This effect is significantlyincreased by addition of an electron donating ligand (push system) ontothe azobenzene aryl ring.

Transition to the cis-geometry upon light exposure will disrupt thiseffect, resulting in increased binding affinity.

In addition, it is important to appreciate that the azobenzene motif ispredicted to bind within the catalytic pocket, which is spatiallyconfined. It is therefore reasonable to expect that one geometry willprovide a better fit compared to the other. Provided that thecis-configuration represents the preferred binding geometry, bothelectronic and spatial contributions will be additive and result in thelargest differential activity between the trans and cis isomer. A set ofcompounds have been prepared which exhibit the benefits and structuralproperties described above.

Provided herein is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof,wherein:X and Y are independently a substituted or unsubstituted aryl orheteroaryl ring, wherein at least one of the rings is substituted withone or more HDAC targeting elements. In some embodiments, the HDACtargeting element is selected from the group consisting of a substitutedor unsubstituted aminobenzamide, a substituted or unsubstitutedhydroxybenzamide, and hydroxamic acids. For example, the HDAC targetingelement can include the terminal phenylformamide moiety of an benzamidehistone deacetylase (HDAC) inhibitor.

In some embodiments, Y is substituted with one or more HDAC targetingelements and X is substituted with one or more fluorescent moieties.Non-limiting examples of suitable fluorescent moieties includeboron-dipyrromethene (BODIPY®),4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid(BODIPY® FL),6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)hexanoicacid, succinimidyl ester (BODIPY® TRM-X), Oregon Green 88,6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoicacid, succinimidyl ester (BODIPY® 650/665-X), a Coumarin, such as7-N,N-diethylaminocoumarin and Coumarin 343, sulforhodamine 101 acidchloride (Texas Red), VIVOTAG 680 (an amine-reactive near-infra-redfluorochrome, from VisEn Medical), umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, and dansyl chloride or phycoerythrin. See, for exampleSAS130.

In some embodiments, a compound of Formula (I) is a compound of Formula(II):

or a pharmaceutically acceptable salt thereof,wherein:

-   each R¹ is independently selected from the group consisting of:    hydrogen, C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkynyl, halo, C₁₋₆ haloalkyl,    CN, NO₂, OR⁴, SR⁴, C(O)R⁴, C(O)NR⁴R⁵, C(O)OR⁴, OC(O)R⁴, OC(O)NR⁴R⁵,    C(═NR⁴)NR⁵R⁶, NR⁴C(═NR⁵)NR⁶R⁷, NR⁴R⁵, NR⁴C(O)R⁵, NR⁴C(O)OR⁵,    NR⁴C(O)NR⁵R⁶, NR⁴S(O)R⁵, NR⁴S(O)₂R⁵, NR⁴S(O)₂NR⁵R⁶, S(O)R⁴,    S(O)NR⁴R⁵, S(O)₂R⁴, S(O)₂NR⁴R⁵, C₁₋₆alkoxyalkyl, carbocyclyl,    C₁₋₆carbocyclylalkyl, heterocyclyl, C₁₋₆heterocyclylalkyl, aryl,    C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl;-   R² is an HDAC targeting element;-   R³ is independently selected from the group consisting of:    C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, halo, C₁₋₆ haloalkyl, CN, NO₂,    OR⁴, SR⁴, C(O)R⁴, C(O)NR⁴R⁵, C(O)OR⁴, OC(O)R⁴, OC(O)NR⁴R⁵,    C(═NR⁴)NR⁵R⁶, NR⁴C(═NR⁵)NR⁶R⁷, NR⁴R⁵, NR⁴C(O)R⁵, NR⁴C(O)OR⁵,    NR⁴C(O)NR⁵R⁶, NR⁴S(O)R⁵, NR⁴S(O)₂R⁵, NR⁴S(O)₂NR⁵R⁶, S(O)R⁴,    S(O)NR⁴R⁵, S(O)₂R⁴, S(O)₂NR⁴R⁵, C₁₋₆alkoxyalkyl, carbocyclyl,    C₁₋₆carbocyclylalkyl, heterocyclyl, C₁₋₆heterocyclylalkyl, aryl,    C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl;-   each R⁴, R⁵, and R⁶ are independently selected from H and C₁₋₆alkyl;-   m is an integer from 0 to 4; and-   n is an integer from 1 to 5.

In some embodiments, a compound of Formula (I) is a compound of Formula(III):

or a pharmaceutically acceptable salt form thereof;wherein:

-   each R¹ is selected from the group consisting of: hydrogen,    C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, halo, C₁₋₆ haloalkyl, CN, NO₂,    OR⁴, SR⁴, C(O)R⁴, C(O)NR⁴R⁵, C(O)OR⁴, OC(O)R⁴, OC(O)NR⁴R⁵,    C(═NR⁴)NR⁵R⁶, NR⁴C(═NR⁵)NR⁶R⁷, NR⁴R⁵, NR⁴C(O)R⁵, NR⁴C(O)OR⁵,    NR⁴C(O)NR⁵R⁶, NR⁴S(O)R⁵, NR⁴S(O)₂R⁵, NR⁴S(O)₂NR⁵R⁶, S(O)R⁴,    S(O)NR⁴R⁵, S(O)₂R⁴, S(O)₂NR⁴R⁵, C₁₋₆alkoxyalkyl, carbocyclyl,    C₁₋₆carbocyclylalkyl, heterocyclyl, C₁₋₆heterocyclylalkyl, aryl,    C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl;-   each R⁴, R⁵, and R⁶ are independently selected from H and C₁₋₆alkyl;-   R² is an HDAC targeting element; and-   n is an integer from 1 to 5.

In some embodiments, R¹ is an electron donating substituent. Essentiallyany functional group capable of releasing electrons into the pi-electronsystem of an aromatic ring system is suitable for use as an electrondonating group, provided that the group is also capable of beingcovalently attached to the aryl ring. An electron donating substituentcan include, for example, a substituent having a Hammett σ_(p) value ofless than zero (see, for example, “A survey of Hammett substituentconstants and resonance and field parameters”, Corwin. Hansch, A. Leo,R. W. Taft Chem. Rev., 1991, 91 (2), pp 165-195).

Examples of suitable electron donating groups known in the art includeNR⁵R⁶, OR⁵, SR⁵, C₁₋₆ alkyl, CH═N—NR⁵R⁶, CH═C(NR⁵R⁶)₂, NR⁵COR⁶,NR⁵C(O)NR⁶R⁷, aryl, and heteroaryl; wherein each R⁵, R⁶ and R⁷ areindependently selected from H and C₁₋₆ alkyl. For example, R¹ can beNR⁵R⁶. In some embodiments, R¹ is N(CH₃)₂.

HDAC targeting elements include any small molecule compound capable ofbinding to HDAC. In some embodiments, the HDAC targeting element isselected from the group consisting of a substituted or unsubstitutedaminobenzamide, a substituted or unsubstituted hydroxybenzamide, andhydroxamic acids. For example, the HDAC targeting element can includethe terminal phenylformamide moiety of an benzamide histone deacetylase(HDAC) inhibitor.

In some embodiments, an HDAC targeting element includes an HDACinhibitor. In some embodiments, suitable histone deacetylase (HDAC)inhibitors can include those compounds having a phenyl benzamide moiety.For example, CI-994, Entinostat (MS-275),

HDAC1/2 Selective CI-994 Analog

(see, e.g., Methot, J. L. et al., Bioorg. Med Chem. Lett., 18(3), 973-8(2008)),

and analogs thereof. See also WO 2004/058234; Methot, J. L. et al.,Bioorg. Med Chem. Lett., 18(3), 973-8 (2008); Witter, D. J. et al.,Bioorg. Med. Chem. Lett., 18(2), 726-31 (2008); and Haggarty, S. J. etal., Neurobiol Learn Mem, 96(1), 41-52 (2011), all of which areincorporated by reference in their entirety herein. Analogs of benzamideHDAC inhibitors can include compounds having an additional substituenton the terminal phenyl ring of the phenyl benzamide moiety. Substituentscan include aryl and heteroaryl rings. For example, the phenyl ring canbe substituted with a phenyl or thienyl moiety in addition to thesubstituents present on the parent HDAC inhibitor. See, for example, theHDAC1/2 selective CI-994 analog shown above.

In some embodiments, the HDAC inhibitor is a HDAC3 selective inhibitors(see, e.g., Haggarty, S. J. et al., Neurobiol Learn Mem, 96(1), 41-52(2011)). HDAC3 selective compounds are characterized by a 4-fluorosubstitutent, such as:

In some embodiments, m is 0. In some embodiments, n is an integer from 1to 2. For example, n can be 1.

Also provided herein is a compound of Formula (IV):

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein:    -   each R¹ is independently an electron donating substituent;    -   each R² is independently selected from the group consisting of:        halogen, NR³R⁴, OR³, aryl, and heteroaryl;    -   each R³ and R⁴ is independently selected from the group        consisting of: H, C₁₋₆ alkyl, and a nitrogen protecting group;    -   m is an integer from 1 to 5; and    -   n is an integer from 1 to 5.

In some embodiments, R¹ is an electron donating substituent as describedabove. For example, R¹ can include: NR⁵R⁶, OR⁵, SR⁵, C₁₋₆ alkyl,CH═N—NR⁵R⁶, CH═C(NR⁵R⁶)₂, NR⁵C(O)NR⁶R⁷, aryl, and heteroaryl; whereineach R⁵, R⁶ and R⁷ are independently selected from H and C₁₋₆ alkyl. Forexample, R¹ can be NR⁵R⁶. In some embodiments, R¹ is N(CH₃)₂. In someembodiments, n is 1 and R¹ is in the para position on the ring.

In some embodiments, R² is NR³R⁴. For example R² can be NH₂. In someembodiments, R² is selected from the group consisting of NH₂, OH,phenyl, and thiophenyl. In some embodiments, m is 1 and R² is in theortho position on the ring. In some embodiments, m is 2 and the first R²is in the ortho position and the second R² is in the meta positionacross the ring from the first R².

As indicated, certain amino groups of the Formula (II) structure may beprotected with an nitrogen protecting group. For this purpose, theprotecting group may include any suitable functional group chosen by aperson skilled in the chemical arts. For example, amino protectinggroups within the scope of the present disclosure include, but are notlimited to, carbamate, amide, A-alkyl, or A-aryl-derived protectinggroups. Each protecting group may be the same or different.

In particular, the carbamate protecting group may include, for example,9-fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc),carboxybenzyl carbamate (cbz), methyl carbamate, ethyl carbamate,9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethylcarbamate, 17-tetrabenzo[a,c,g,i]fluorenylmethyl carbamate (Tbfmoc),2-chloro-3-indenylmethyl carbamate (Climoc),2,7-di-t-butyl[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 1,1-dioxobenzo[b]thiophene-2-ylmethyl carbamate(Bsmoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethylcarbamate (Teoc), 2-phenylethyl carbamate (hZ), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-boc),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBoc),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (7-Bumeoc),N-2-pivaloylamino)-1,1-dimethylethyl carbamate,2-[(2-nitrophenyl)dithio]-1-phenylethyl carbamate (NpSSPeoc),2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, 1-adamantyl carbamate(1-Adoc), vinyl carbamate (Voc), 1-isopropylallyl carbamate (Ipaoc),4-nitrocinnamyl carbamate (Noc), 3-(3′pyridyl)prop-2-enyl carbamate(Paloc), 8-quinolyl carbamate, alkyldithio carbamate, p-methoxybenzylcarbamate (Moz), p-nitrobenzyl carbamate (Pnz), p-bromobenzyl carbamate,p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate,4-methylsulfinylbenzyl carbamate (Msz), diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonoethyl carbamate(Peoc), 1,1-dimethyl-2-cyanoethyl carbamate, 2-(4-nitrophenyl)ethylcarbamate, 4-phenylacetoxybenzyl carbamate (PhAcOZ), andm-chloro-p-acyloxybenzyl carbamate. In particular, 9-fluorenylmethylcarbamate (Fmoc), t-butyl carbamate (Boc), and carboxybenzyl carbamate(cbz) protecting groups may be used.

The amide protecting group may include, for example, acetamide,phenylacetamide, 3-phenylpropanamide, pent-4-enamide, picolinamide,3-pyridylcarboxamide, benzamide, p-phenylbenzamide,2-methyl-2-(o-phenylazophenoxy)propanamide), 4-chlorobutanamide,acetoacetamide, 3-(p-hydroxyphenyl)propanamide), and(N′-dithiobenzyloxycarbonylamino)acetamide.

In some embodiments, the nitrogen protecting group is t-butyl carbamate(Boc).

In some embodiments, m is an integer from 1 to 3 (e.g., 1, 2 or 3). Insome embodiments, m is 1 or 2. For example, m can be 1. In someembodiments, n is an integer from 1 to 3 (e.g., 1, 2 or 3). In someembodiments, n is 1 or 2. For example, n can be 1.

Further provided herein are compounds of Formula (V):

or a pharmaceutically acceptable salt thereof,wherein:

-   R¹ is an electron donating substituent;-   R² is selected from the group consisting of: NR¹⁰R¹¹ and OR¹⁰;-   each R³ is independently selected from the group consisting of:    hydrogen, C₁₋₉alkyl, C₂₋₉alkenyl, C₂₋₉alkynyl, halo, C₁₋₉ haloalkyl,    CN, NO₂, OR⁷, SR⁷, C(O)R⁷, C(O)NR⁷R⁸, C(O)OR⁷, OC(O)R⁷, OC(O)NR⁷R⁸,    C(═NR⁷)NR⁸R⁹, NR⁷C(═NR⁸)NR⁹R⁹, NR⁷R⁸, NR⁷C(O)R⁸, NR⁷C(O)OR⁸,    NR⁷C(O)NR⁸R⁹, NR⁷S(O)R⁸, NR⁷S(O)₂R⁸, NR⁷S(O)₂NR⁸R⁹, S(O)R⁷,    S(O)NR⁷R⁸, S(O)₂R⁷, S(O)₂NR⁷R⁸, C₁₋₉alkoxyalkyl, carbocyclyl,    C₁₋₉carbocyclylalkyl, heterocyclyl, C₁₋₉heterocyclylalkyl, aryl,    C₁₋₉aralkyl, heteroaryl, and C₁₋₉heteroaralkyl;-   each R⁴ is independently selected from the group consisting of: H,    halogen, aryl, and heteroaryl;-   each R⁷, R⁸, and R⁹ is independently selected from the group    consisting of: H, C₁₋₆ alkyl;-   R¹⁰ and R¹¹ are independently selected from the group consisting of:    H, C₁₋₆ alkyl, and a nitrogen protecting group; and-   p is an integer from 0 to 4.

The electron donating substituent and nitrogen protecting group are asdefined above.

In some embodiments, R¹ can be NR⁵R⁶. For example, R¹ can be N(CH₃)₂. Insome such embodiments, p is 0.

In some embodiments, R² is NR¹⁰R¹¹. For example R² can be NH₂. In somesuch embodiments, R⁴ is selected from the group consisting of phenyl,and thiophenyl.

Non-limiting examples of the compounds provided herein include:

and pharmaceutically acceptable salt forms thereof.

Also provided herein are the cis-isomers of the compounds providedabove. Such compounds can be prepared by irradiating the correspondingtrans-isomer with a suitable wavelength of light to induce theconformational change. Accordingly, provided herein are compounds offormula (VI):

or a pharmaceutically acceptable salt thereof,wherein:X and Y are independently a substituted or unsubstituted aryl orheteroaryl ring, wherein at least one of the rings is substituted withone or more HDAC targeting elements. In some embodiments, the HDACtargeting element is selected from the group consisting of a substitutedor unsubstituted aminobenzamide, a substituted or unsubstitutedhydroxybenzamide, and hydroxamic acids. For example, the HDAC targetingelement can include the terminal phenylformamide moiety of an benzamidehistone deacetylase (HDAC) inhibitor.

Also provided herein is a compound of Formula (VII):

or a pharmaceutically acceptable salt thereof,wherein:

-   each R¹ is independently selected from the group consisting of:    hydrogen, C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, halo, C₁₋₆ haloalkyl,    CN, NO₂, OR⁴, SR⁴, C(O)R⁴, C(O)NR⁴R⁵, C(O)OR⁴, OC(O)R⁴, OC(O)NR⁴R⁵,    C(═NR⁴)NR⁵R⁶, NR⁴C(═NR⁵)NR⁶R⁷, NR⁴R⁵, NR⁴C(O)R⁵, NR⁴C(O)OR⁵,    NR⁴C(O)NR⁵R⁶, NR⁴S(O)R⁵, NR⁴S(O)₂R⁵, NR⁴S(O)₂NR⁵R⁶, S(O)R⁴,    S(O)NR⁴R⁵, S(O)₂R⁴, S(O)₂NR⁴R⁵, C₁₋₆alkoxyalkyl, carbocyclyl,    C₁₋₆carbocyclylalkyl, heterocyclyl, C₁₋₆heterocyclylalkyl, aryl,    C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl;-   R² is an HDAC targeting element;-   R³ is independently selected from the group consisting of:    C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, halo, C₁₋₆ haloalkyl, CN, NO₂,    OR⁴, SR⁴, C(O)R⁴, C(O)NR⁴R⁵, C(O)OR⁴, OC(O)R⁴, OC(O)NR⁴R⁵,    C(═NR⁴)NR⁵R⁶, NR⁴C(═NR⁵)NR⁶R⁷, NR⁴R⁵, NR⁴C(O)R⁵, NR⁴C(O)OR⁵,    NR⁴C(O)NR⁵R⁶, NR⁴S(O)R⁵, NR⁴S(O)₂R⁵, NR⁴S(O)₂NR⁵R⁶, S(O)R⁴,    S(O)NR⁴R⁵, S(O)₂R⁴, S(O)₂NR⁴R⁵, C₁₋₆alkoxyalkyl, carbocyclyl,    C₁₋₆carbocyclylalkyl, heterocyclyl, C₁₋₆heterocyclylalkyl, aryl,    C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl;-   each R⁴, R⁵, and R⁶ are independently selected from H and C₁₋₆alkyl;-   m is an integer from 0 to 4; and-   n is an integer from 1 to 5.

In some embodiments, a compound of Formula (VI) is a compound of Formula(VIII):

or a pharmaceutically acceptable salt thereof,wherein:

-   each R¹ is selected from the group consisting of: hydrogen,    C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, halo, C₁₋₆ haloalkyl, CN, NO₂,    OR⁴, SR⁴, C(O)R⁴, C(O)NR⁴R⁵, C(O)OR⁴, OC(O)R⁴, OC(O)NR⁴R⁵,    C(═NR⁴)NR⁵R⁶, NR⁴C(═NR⁵)NR⁶R⁷, NR⁴R⁵, NR⁴C(O)R⁵, NR⁴C(O)OR⁵,    NR⁴C(O)NR⁵R⁶, NR⁴S(O)R⁵, NR⁴S(O)₂R⁵, NR⁴S(O)₂NR⁵R⁶, S(O)R⁴,    S(O)NR⁴R⁵, S(O)₂R⁴, S(O)₂NR⁴R⁵, C₁₋₆alkoxyalkyl, carbocyclyl,    C₁₋₆carbocyclylalkyl, heterocyclyl, C₁₋₆heterocyclylalkyl, aryl,    C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl;-   each R⁴, R⁵, and R⁶ are independently selected from H and C₁₋₆alkyl;-   R² is an HDAC targeting element; and-   n is an integer from 1 to 5.

Further provided herein are compounds of Formula (IX):

or a pharmaceutically acceptable salt thereof, wherein each R¹ isindependently an electron donating substituent; each R² is independentlyselected from the group consisting of: halogen, NR³R⁴, OR³, aryl, andheteroaryl; each R³ and R⁴ is independently selected from the groupconsisting of: H, C₁₋₆ alkyl, and a nitrogen protecting group; m is aninteger from 1 to 5; and n is an integer from 1 to 5 as defined abovefor the compounds of Formula (II).

Finally, provided herein are compounds of Formula (X):

or a pharmaceutically acceptable salt thereof, wherein: wherein:

-   R¹ is an electron donating substituent;-   R² is selected from the group consisting of: NR¹⁰R¹¹ and OR¹⁰;-   each R³ is independently selected from the group consisting of:    hydrogen, C₁₋₉alkyl, C₂₋₉alkenyl, C₂₋₉alkynyl, halo, C₁₋₉ haloalkyl,    CN, NO₂, OR⁷, SR⁷, C(O)R⁷, C(O)NR⁷R⁸, C(O)OR⁷, OC(O)R⁷, OC(O)NR⁷R⁸,    C(═NR⁷)NR⁸R⁹, NR⁷C(═NR⁸)NR⁹R⁹, NR⁷R⁸, NR⁷C(O)R⁸, NR⁷C(O)OR⁸,    NR⁷C(O)NR⁸R⁹, NR⁷S(O)R⁸, NR⁷S(O)₂R⁸, NR⁷S(O)₂NR⁸R⁹, S(O)R⁷,    S(O)NR⁷R⁸, S(O)₂R⁷, S(O)₂NR⁷R⁸, C₁₋₉alkoxyalkyl, carbocyclyl,    C₁₋₉carbocyclylalkyl, heterocyclyl, C₁₋₉heterocyclylalkyl, aryl,    C₁₋₉heteroaryl, and C₁₋₉heteroaralkyl;-   each R⁴ is independently selected from the group consisting of: H,    halogen, aryl, and heteroaryl;-   each R⁷, R⁸, and R⁹ is independently selected from the group    consisting of: H, C₁₋₆ alkyl;-   R¹⁰ and R¹¹ are independently selected from the group consisting of:    H, C₁₋₆ alkyl, and a nitrogen protecting group; and-   p is an integer from 0 to 4.    Pharmaceutical Compositions and Methods of Administration

The methods described herein include the manufacture and use ofpharmaceutical compositions, which include the compounds provided hereinas active ingredients. Also included are the pharmaceutical compositionsthemselves.

Pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier. A pharmaceutically acceptable carrier includessaline, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration.

Methods of formulating suitable pharmaceutical compositions are known inthe art, see, e.g., Remington: The Science and Practice of Pharmacy,21st ed., 2005; and the books in the series Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs (Dekker, NY). Forexample, solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of a therapeutic compound as described hereincan be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays or suppositories. For transdermal administration, the activecompounds are formulated into ointments, salves, gels, or creams asgenerally known in the art.

The pharmaceutical compositions can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

The term “ophthalmic composition” as used herein will be understood torefer to any composition for direct or local administration to an eye ofa patient. The composition may be administered topically to the eye ormay be injected into the eye (e.g., intravitreal injection,subconjunctival injection, sub-tenon injection, retrobulbar injection,subretinal injection, suprachoroidal injection, and the like). Theophthalmic composition may be provided in any form that allows local ordirect administration thereof to the eye, including but not limited to,a solution, drops, mist/spray, plasters and pressure sensitiveadhesives, ointment, lotion, cream, gel, lyophilized/spray-dried forms,rods, beads, emulsions, lenses, patch, plug, elixir, and the like. Theophthalmic compositions provided herein typically vary according to theparticular active agent (i.e., a compound provided herein) used, thepreferred drug release profile, the condition being treated, and themedical history of the patient. In addition, the ophthalmic compositionsof the present disclosure may be designed to provide delayed, controlledor sustained release using formulation techniques which are well knownin the art.

Any of the ophthalmic compositions described and claimed herein mayfurther comprise at least one delivery agent that assists in thepenetration of a surface of an eye; in certain embodiments, the deliveryagent may assist in delivery to the cornea and/or retina of the eye. Forexample, in order for a topical application to be effective, thecomposition may need to be able to penetrate the surface of the eye sothat it can travel to the desired tissue. This may include penetratingthe conjunctiva and/or the cornea. Also, the penetration rate must besufficient to impart an effective dose. Many drugs do not possess arequisite penetration ability with regard to the tissues of the eye. Itshould be noted that the external layers of the eye are quite differentfrom the tissues encountered in the stomach and intestinal tract. Thus,while a certain drug may be readily absorbed in the intestines andintroduced into the blood supply for systemic administration, the samedrug may be incapable of being absorbed by or passing through thesubstantially avascular outer layers of the conjunctiva or cornea at aminimally acceptable therapeutic concentration. The mechanism oftransport or uptake of the drug is entirely different for topicaladministration than for oral administration.

In one embodiment, the compounds provided herein are prepared withcarriers that will protect the compounds against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Suchformulations can be prepared using standard techniques, or obtainedcommercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions (including liposomes targeted to selected cellswith monoclonal antibodies to cellular antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. For example, a therapeutically effective amount is onethat achieves the desired therapeutic effect. This amount can be thesame or different from a prophylactically effective amount, which is anamount necessary to prevent onset of disease or disease symptoms. Aneffective amount can be administered in one or more administrations,applications or dosages. A therapeutically effective amount of atherapeutic compound (i.e., an effective dosage) depends on thecompounds selected. The compositions can be administered from one ormore times per day to one or more times per week; including once everyother day. The skilled artisan will appreciate that certain factors mayinfluence the dosage and timing required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of a compound provided herein caninclude a single treatment or a series of treatments.

Dosage, toxicity and therapeutic efficacy of the compounds providedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit hightherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Methods of Treatment

The methods described herein include methods for the treatment ofdisorders associated with aberrant histone deacetylase activity. In someembodiments, the disorder is cancer. Generally, the methods includeadministering a therapeutically effective amount of a compound asdescribed herein, to a patient who is in need thereof, or who has beendetermined to be in need of, such treatment. Following administration ofthe compound to the patient, the compound can be activated by exposureof the patient to a suitable wavelength of light to convert the compoundfrom its inactive or relatively inactive trans isomer to the active ormore active cis isomer.

The compounds and compositions provided herein can be used as inhibitorsand/or modulators of histone deacetylases, and thus can be used to treata variety of disorders and diseases in which histone deacetylaseactivity is implicated, such as proliferative diseases (e.g., cancers,benign neoplasms, angiogenesis, inflammatory diseases, and autoimmunediseases, genetic diseases).

In certain embodiments, the disease can be proliferative diseases, suchas cancer; autoimmune diseases; allergic and inflammatory diseases;diseases of the central nervous system (CNS), such as neurodegenerativediseases (e.g., Huntington's disease, amyotrophic lateral sclerosis(ALS)); vascular diseases, such as restenosis; musculoskeletal diseases;cardiovascular diseases, such as stroke; pulmonary diseases; gastricdiseases; genetic diseases, such as spinal muscle atrophy; infectiousdiseases; diseases associated with an HPV infection; and Alzheimer'sdisease.

Histone deacetylase is known to play an essential role in thetranscriptional machinery for regulating gene expression, induce histonehyperacetylation and to affect the gene expression. Therefore, it isuseful as a therapeutic or prophylactic agent for diseases caused byabnormal gene expression such as inflammatory disorders, diabetes,diabetic complications, homozygous thalassemia, fibrosis, cirrhosis,acute promyelocytic leukaemia (APL), organ transplant rejections,autoimmune diseases, protozoal infections, and tumors.

In some embodiments, a compound or composition provided herein can beused to treat cancer (e.g., skin cancer) and/or retinal disorders.Accordingly, the compounds and compositions provided herein can be usedto treat cancer and/or retinal disorders. Examples of diseases which canbe treated with the compounds and compositions provided herein include avariety of cancers, including skin cancer, and retinal disordersincluding, for example, ischemic retinal injury, and retinaldegeneration (e.g., retinitis pigmentosa).

With respect to cancer, histone deacetylase is thought to be involved inthe regulation of cellular proliferation in a variety of cancersincluding, for example, skin cancer. Non-limiting examples of skincancers include malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, and scleroderma.

Other cancers can also be treated with the compounds and compositionsdescribed herein.

A method of treating cancer using a compound or composition as describedherein may be combined with existing methods of treating cancers, forexample by chemotherapy, irradiation, or surgery (e.g., removal ofskin). In some embodiments, a compound or composition can beadministered before, during, or after another anticancer agent ortreatment.

A method of inhibiting an HDAC in a cell is also provided herein, themethod comprising contacting the cell with an effective amount of acompound of Formula (I). In some embodiments, the method furthercomprising exposing the cell to a light suitable to convert the compoundof Formula (I) to its cis confirmation. The method of inhibiting an HDACin a cell may be performed by contacting the cell with a compoundaccording to Formula (I), or a pharmaceutically acceptable salt formthereof, in vitro, thereby inducing inhibition of an HDAC of a cell invitro. Uses of such an in vitro method of inhibiting an HDAC include,but are not limited to use in a screening assay (for example, wherein acompound according to Formula (I) is used as a positive control orstandard compared to compounds of unknown activity or potency ininhibiting HDAC). In some embodiments thereof, HDAC is inhibited in acancer cell (e.g., a skin cancer cell).

The method of inhibiting an HDAC in a cell may be performed, forexample, by contacting a cell with a compound according to Formula (I),in vivo, thereby inhibiting an HDAC in a subject in vivo. The contactingis achieved by causing a compound according to Formula (I), or apharmaceutically acceptable salt form thereof, to be present in thesubject in an amount effective to achieve inhibition of the HDAC. Thismay be achieved, for example, by administering an effective amount of acompound according to Formula (I), or a pharmaceutically acceptable saltform thereof, to a patient. In some embodiments, the method furthercomprising exposing the cell to a light suitable to convert the compoundof Formula (I) to its cis confirmation. Such exposure can occur, forexample, by exposing the patient to the light (e.g., exposing theportion of the patient wherein the cell of interest is located). Uses ofsuch an in vivo method of inhibiting HDAC include, but are not limitedto, use in methods of treating a disease or condition, whereininhibiting HDAC is beneficial. In some embodiments thereof, the HDAC isinhibited in a cancer cell, for example in a patient suffering fromcancer. The method can be performed by administering an effective amountof a compound according to Formula (I), or a pharmaceutically acceptablesalt form thereof, to a patient who is suffering from cancer.

Without being bound by theory, when Y is substituted with an HDACtargeting element and X is substituted with a fluorescent moiety, it isbelieved that the portion of the compound of Formula (I) including theHDAC targeting element can function as a fluorescent quencher while thecompound of Formula (I) is in its unbound or trans configuration, thusquenching the fluorescent signal produced by the compound. The portionof the compound including the HDAC targeting element functions similarlyto DABCYL. Upon conversion to the cis configuration and subsequentbinding of the compound to the active site of HDAC, the DABCYL-likemoiety has a decreased ability to quench the fluorescence of thefluorescent moiety and the fluorescent signal produced by the compoundcan increase significantly. Accordingly, in some embodiments, a compoundprovided herein can be used in a method to detectably label an HDAC(e.g., the active site of an HDAC) in a cell or in a patient. Thefluorescent signal produced by the bound compound can be detected bymethods known by those of skill in the art.

As is discussed above, the compounds provided herein are administered inthe inactive or less active trans configuration. Followingadministration of the compound to the patient, the compound can beactivated by exposure of the patient to light, thus converting thecompound from the inactive trans to the active cis configuration. Insome embodiments, the light can be targeted specifically at the area ofthe patient to be treated. For example, light can be focused onto thecancerous portions of the skin or into the retina of the eye. In thisway, the activated drug is able to bind and inhibit HDAC at the locationof need while much of the remaining inactive drug is removed from thepatient through normal excretion pathways. In some embodiments, thethermal relaxation of the compound from the active or more active cisconfiguration to the trans configuration is fast and upon removal of thepatient from the light source, any unbound compound thermally relaxesback into the inactive or less active state.

Any suitable light source may be used to activate the compounds providedherein. For example, topical and intraocular application of visiblelight; a laser can be used to provide light to the patient; for exposureof the blood to light, the light can be administered from outside of thebody or intravenously. In some embodiments, after the location of cancercells is determined, laser energy of a desired wavelength, intensity,duration and modulation is delivered to the cancer cells. In someembodiments, after the location of the area to be treated is determined(e.g., the eye), laser energy of a desired wavelength, intensity,duration and modulation is delivered to the targeted location.

Delivery of light may be by, for example, light bulb, LED, fluorescentlight tube, sunlight, and/or direct laser application to the affectedregion of the body. An example of wavelengths of light effective toconvert the compounds provided herein from the trans to cisconfiguration are from about 200 nm to about 800 nm. In someembodiments, the wavelength of light ranges from about 350 nm to about550 nm. For example, the light can be in the blue region of the visiblespectrum (i.e., about 450 nm to about 495 nm). The energy level of thelight may be from 0.1 watt to 15 watts. In some embodiments, the energylevel of the light is less than 0.1 watt. An example treatment time forexposing the patient to light can be from less than 1 minute to morethan 1 hour.

In some embodiments, direct laser application of light is made to theaffected region of the body. Alternatively, a fiber needle or fiber canbe used to deliver laser light/laser energy to the patient. For example,a fiber needle assembly to delivery laser energy to targeted locationcan be one or multiple fibers depending on the size of the location(e.g., depending on the size of the tumor to be treated). Multiple fiberneedles can be inserted inside the body in different directions so thatthe targeted location can be surrounded or covered completely by laserenergy coming at the location from different directions.

Lasers can be solid state lasers, gas lasers, semiconductor lasers andothers. An example of wavelengths of laser light effective to convertthe compounds provided herein from the trans to cis configuration arefrom about 200 nm to about 800 nm. In some embodiments, the wavelengthof light ranged from about 350 nm to about 550 nm. For example, thelight can be in the blue region of the visible spectrum (i.e., about 450nm to about 495 nm). The energy level of a laser may be from 0.1 watt to15 watts. In some embodiments, the energy level of the laser is lessthan 0.1 watt. An example treatment time for exposing the patient tolaser energy can be from less than 1 minute to more than 1 hour. Thelaser energy applied to the patient may also be modulated. Laser energymay be applied to the targeted location by continuous wave (constantlevel), pulsing (on/off), ramping (from low to high power levels, orfrom high to low power levels), or other waveform (such as sine wave,square wave, triangular wave, etc.). Modulation of laser energy may beachieved by modulating power to the laser light source, or by blockingor reducing light output from the laser light source according to adesired modulation pattern.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1—Preparation of Compound Intermediates

Synthesis of 4-bromo-2-nitrophenyl acetate (4)

4-bromo-2-nitrophenyl acetate was made following the reported procedurewith little modification (WO 2005/030705). A solution of4-bromo-2-nitrophenol (0.25 g, 1.147 mmol) in acetic anhydride (2.5 mL)was exposed to microwave reaction at 160° C. for 10 minutes. Most of thesolvent was evaporated in vacuo and the resulting oil was kept in thefreezer for 3 days. Crystallization occurred while thawing the sample atroom temperature. The yellowish crystals were suspended in a mixture ofEtOAc/hexane (9:1) and collected by filtration to afford the titlecompound 4 (0.24 g, 81.1% yield). ¹H NMR: (400 MHz, CDCl3) δ (ppm): 8.22(d, J=2 Hz, 1H), 7.76 (dd, J=5.4 Hz, 2.4 Hz, 1H), 7.13 (d, J=8.4 Hz,1H), 2.358 (s, 3H).

Synthesis of 2-nitro-4-(thiophen-2-yl)phenol (5)

In a microwave vessel, 4-bromo-2-nitrophenyl acetate (1.3 g, 5 mmol),thiophen-2-ylboronic acid (1.14 g, 8.9 mmol), tribasic potassiumphosphate (2.52 g, 11.9 mmol), tris-(dibenzylideneacetone)dipalladium(O)(0.0563 g, 0.061 mmol), and2-dicyclohexyl-phosphino-2′,4′,6′-triisopropyl biphenyl (0.114 bg, 0.239mmol) were added in w-butanol (18 mL) and after degassing with argonexposed to microwave reaction for 20 min. After cooling, the mixture wasdiluted with EtOAc and extracted from water. The organic layer wasrinsed with brine, dried over sodium sulphate and concentrated to give acrude mixture which was then purified by column chromatography usingEtOAc/hexane (20:80) to give the title compound (0.024 g, 22% yield). ¹HNMR: (400 MHz, CDCl₃) δ (ppm): 7.75 (d, J=16 Hz, 1H), 7.6 (m, 2H), 7.4(m, 2H), 7.1 (d, J=16 Hz, 1H).

Synthesis of 2-amino-4-(thiophen-2-yl)phenol (6)

The nitro compound (0.075 g, 0.34 mmol) was hydrogenated via balloon inthe presence of 10% palladium on charcoal (catalytic amount) in methanol(2 mL) at room temperature for 2 hours. The crude product was filteredthrough a pad of Celite, and the filtrate was evaporated to give thedesired compound sufficiently pure to use for the next reaction (0.065g, 100% yield). ¹H NMR: (400 MHz, DMSO-d₆) δ (ppm): 7.34 (d, J=6 Hz,1H), 7.16 (d, J=4.8, 1H), 7.03 (t, J=4.2, 1H), 6.86 (s, 1H), 6.67 (m,2H).

Synthesis of tert-butyl (4-bromo-2-nitrophenyl)carbamate (7)

This intermediate compound was made following the reported procedure (WO2009/055917). To a stirred solution of 4-bromo-2-nitroanile (10.0 g,46.1 mmol) and di-tert-butyl dicarbonate (Boc anhydride) (20.11 g, 92.2mmol) in 100 mL THF was added a catalytic amount of4-(dimethylamino)pyridine (DMAP). The reaction mixture was allowed tostir for 90 minutes, and then the solvent was evaporated in vacuo toyield a thick oil. The oil was dissolved in THF (46 mL) and heated to65° C. for 18 hours. Solid sodium hydroxide (1.8 g, 46.1 mmol) was addedto the reaction mixture and heating was continued for 4 hours; then theTHF was evaporated. The yellow colored solid was dissolved in EtOAc,washed with water, and the organic layer was evaporated and purified bycolumn chromatography using EtOAc/hexane (10:90) to yield the desiredproduct (11 g, 75% yield). ¹H NMR: (400 MHz, DMSO-d₆) δ (ppm): 9.62 (s,1H), 7.98 (d, J=2.2 Hz, 1H), 7.76 (dd, J=8.6, 2.2 Hz, 1H), 7.55 (d,J=8.8 Hz, 1H), 1.44 (s, 9H).

Synthesis of tert-butyl (2-nitro-4-(thiophen-2-yl)phenyl)carbamate (8a)

This compound was synthesized following the reported procedure withlittle modification (WO 2009/055917). A suspension of 2-thiopheneboronic acid (0.22 g, 0.694 mmol), bromoarene 7 (0.118 g, 0.922 mmol),tri-o-tolyl-phosphine (0.069 g, 0.22 mmol), and potassium carbonate(0.288 g, 2.082 mmol) in degassed dimethoxyethane (DME) (1.8 mL) andwater (0.6 mL) was treated with tetrakis (triphenylphosphine)(0) (0.052g, 0.045 mmol). The reaction mixture was exposed to microwave at 80° C.for 30 minutes. After cooling, the reaction mixture was diluted withethyl acetate, washed with brine, dried over MgSO₄ and concentrated. Thecrude material was purified with column chromatography (10% EtOAc inhexane) to give the title compound (0.18 g, 81% yield). ¹H NMR: (400MHz, DMSO-d₆) δ (ppm): 9.59 (s, 1H), 8.10 (d, J=2.2 Hz, 1H), 7.91 (dd,J=8.4, 2.2 Hz, 1H), 7.61-7.58 (m, 2H), 7.57-7.53 (m, 1H), 7.13 (dd,J=4.8, 3.2 Hz, 1H), 1.43 (s, 9H).

Synthesis of tert-butyl (3-nitro-[1,1′-biphenyl]-4-yl)carbamate (8b)

That compound was synthesized following the same procedure as describedfor 8a. The suspension of phenyl boronic acid (0.865 g, 7.1 mmol),bromoarene 7 (1.5 g, 4.7 mmol), tri-o-tolyl-phosphine (0.446 g, 1.5mmol), and potassium carbonate (1.96 g, 142 mmol) in degasseddimethoxyethane (DME) (9 mL) and water (3 mL) was treated withtetrakis(triphenylphosphine)(0) (0.410 g, 0.36 mol). The reactionmixture was exposed to microwave at 80° C. for 30 minutes. Aftercooling, the reaction mixture was diluted with ethyl acetate, washedwith brine, dried over MgSO₄, and concentrated. The crude material waspurified with column chromatography (5% EtOAc in hexane) to give thetitle compound (1.085 g, 72.4% yield). ¹H NMR (400 MHz, Chloroform-d) δ9.67 (s, 1H), 8.63 (d, J=8.9 Hz, 1H), 8.42 (s, 1H), 7.87-7.82 (m, 1H),7.62-7.57 (m, 2H), 7.50-7.44 (m, 2H), 7.42-7.37 (m, 1H), 1.56 (s, 9H).

Synthesis of tert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate (9a)

Compound 9a was made following the procedure reported in the patentliterature (WO 2009/055917). Compound 8a (0.24 g, 0.75 mmol) was placedin a round bottom flask and 10% palladium on carbon (catalytic amount,20 mg) was added into it. The reaction mixture was exposed to a H₂balloon. After purging with H₂, the reaction mixture was stirred underH₂ balloon for 2 hours at room temperature. It was then filtered throughCelite and concentrated under vacuum to give the desired amine (0.217 g,100% yield). ¹H NMR (400 MHz, Chloroform-d) δ 7.33-7.27 (m, 1H),7.25-7.21 (m, 2H), 7.08-7.03 (m, 2H), 7.02 (d, J=2.0 Hz, 1H), 1.52 (s,9H).

Synthesis of tert-butyl (3-amino-[1,1′-biphenyl]-4-yl)carbamate (9b)

Compound 9b was made following the same procedure described for 9a.Synthesized intermediate 8a (0.27 g, 0.86 mmol) was used to give aquantitative yield (0.244 g) of title compound 9b. ¹H NMR: (400 MHz,CDCl₃) δ (ppm): ¹H NMR (400 MHz, Chloroform-d) δ 7.55-7.51 (m, 2H), 7.40(dd, J=8.2, 6.7 Hz, 2H), 7.34-7.31 (m, 2H), 7.04 (m, 1H), 6.99 (d, J=2.0Hz, 1H), 1.53 (s, 9H).

Example 2—Preparation of Compounds

General Procedure Followed for the Aromatic Acid and Amine CouplingReactions (A);

This procedure was followed for aromatic acid and aromatic aminecoupling reactions where the aromatic acids containing unsubstituted or4′ substituted diphenyl-diazene moiety were suspended indichloromethane/pyridine (1:1) mixture and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDCI) was added into itand stirred for 10 minutes. To this stirring solution, amines and acatalytic amount of 4-DMAP were added at room temperature and stirringwas continued to 2 hours. The reaction mixture was evaporated and thecrude mixture was resuspended into ethyl acetate and extracted from anaqueous NaHCO₃ solution. After evaporating the EtOAc layer, the titlecompounds were purified by column chromatography using ethyl acetatehexane mixture that ratio was determined from analytical silicagel-coated TLC plates (Silica Gel 60 F₂₅₄).

Synthesis of N-(2-aminophenyl)-4-(phenyldiazenyl)benzamide (10a)

Compound 10a was synthesized following the general acid-amine couplingreaction (A) described above where 4-(phenylazo)benzoic acid (0.2 g,0.884 mmol) was treated with o-phenylenediamine (0.287 g, 2.65 mmol) toyield final compound 10a (0.23 g, 82% yield). ¹H NMR: (400 MHz, DMSO-d₆)δ (ppm): 9.83 (s, 1H), 8.19 (d, J=7.2 Hz, 2H), 7.98 (d, J=7.2 Hz, 2H),7.93 (d, J=6 Hz, 2H), 7.59 (m, 3H), 7.17 (d, J=7.2 Hz, 1H), 6.98 (t,J=6.8 Hz, 1H), 6.78 (d, J=7.6, 1H), 6.6 (t, J=6.8 Hz, 1H). ¹³C NMR (101MHz, dmso) δ 164.99, 153.79, 152.35, 143.74, 137.30, 132.50, 130.02,129.59, 127.26, 127.12, 123.44, 123.22, 122.75, 116.62, 116.51.

Synthesis ofN-(2-aminophenyl)-4-((4-(dimethylamino)phenyl)diazenyl)benzamide (10b)

Compound 10b was made following the same procedure described for 10a.4-Dimethylamino azobenzene-4′-carboxylic acid (0.25 g, 0.93 mmol) wastreated with o-phenylenediamine (0.301 g, 2.78 mmol) to give the finalcompound 10b (0.247 g, 74% yield). ¹H NMR: (400 MHz, DMSO-d₆) δ (ppm):9.8 (s, 1H), 8.12 (d, J=8 Hz, 2H), 7.86 (t, J=9 Hz, 4H), 7.17 (d, J=7.6Hz, 1H), 6.99 (t, J=7.2 Hz, 1H), 6.85 (d, J=9.2 Hz, 2H), 6.79 (d, J=8Hz, 1H), 6.62 (t, J=7.6 Hz, 1H), 4.91 (s, 2H), 3.06 (s, 6H). ¹³C NMR(101 MHz, dmso) δ 165.15, 154.54, 153.30, 143.68, 143.07, 135.28,129.41, 127.20, 127.00, 125.59, 123.66, 121.92, 116.66, 116.54, 112.02.

Synthesis of4-((4-(dimethylamino)phenyl)diazenyl)-N-(2-hydroxyphenyl)benzamides(10c)

Compound 10c was also made following the general procedure A. To asuspension of 4-dimethylamino-azobenzene-4′-carboxylic acid (0.308 g,1.145 mmol), 2-aminophenol (0.05 g, 0.458 mmol) was added. After thecompletion of the reaction, the solution was evaporated and resuspendedin dichloromethane and then washed with water and neutralized with 0.1(N) aqueous NaOH. The organic layer was evaporated and the crude mixturewas purified by column chromatography using 5% methanol in ethyl acetateto yield the desired compound 10c (0.142 g, 86% yield). ¹H NMR (400 MHz,DMSO-d6) δ 9.73 (s, 1H), 9.61 (s, 1H), 8.10 (d, J=8.6 Hz, 2H), 7.89-7.73(m, 4H), 7.71-7.62 (m, 1H), 6.93 (d, J=1.4 Hz, 1H), 6.84 (dd, J=9.4, 2.2Hz, 3H), 3.07 (s, 6H). ¹³C NMR (101 MHz, dmso) δ 165.09, 154.69, 153.34,150.00, 143.08, 134.95, 129.20, 126.28, 126.21, 125.63, 124.86, 122.11,119.46, 116.46, 112.03.

Synthesis of 4-((4-((2-aminophenyl)carbamoyl)phenyl)diazenyl)benzoicAcid (10d)

This compound was made following the procedure A whereo-phenylenediamine (0.04 g, 0.37 mmol) and azobenzene-4,4′-dicarboxylicacid (0.10 g, 0.37 mmol) were used to get the title compound 10d (0.025g, 19% yield). ¹H NMR: (400 MHz, DMSO-d₆) δ (ppm): 8.21-8.19 (d, J=8 Hz,1H), 8.14-8.12 (m, 2H), 8.02-8.00 (d, J=8 Hz, 1H), 7.97-7.93 (m, 3H),7.82-7.80 (d, J=8 Hz, 1H), 7.19-7.17 (d, J=8 Hz, 1H), 6.99-6.92 (m, 2H),6.79-6.76 (d, J=12 Hz, 1H), 6.09-6.00 (m, 1H). ¹³C NMR (101 MHz, dmso) δ172.48, 167.51, 164.98, 154.06, 153.79, 143.74, 137.64, 130.92, 130.54,129.63, 127.97, 127.27, 123.41, 122.96, 122.93, 120.11, 116.59, 116.49.

Synthesis of4-((4-(dimethylamino)phenyl)diazenyl)-N-(2-hydroxyphenyl)benzamides(10e)

This compound was synthesized following the same procedure used to makecompound 10c. 4-Dimethylamino-azobenzene-4′-carboxylic acid (0.211 g,0.784 mmol) and 2-amino-4-(thiophen-2-yl)phenol were exposed to thecoupling reaction to yield the desired final compound 10e (0.73 g, 65%yield). ¹H NMR (400 MHz, DMSO-d6) δ 8.16-8.04 (m, 2H), 8.00 (d, J=2.3Hz, 1H), 7.90-7.77 (m, 4H), 7.43 (dd, J=5.1, 1.2 Hz, 1H), 7.39-7.28 (m,2H), 7.08 (dd, J=5.1, 3.5 Hz, 1H), 6.96 (d, J=8.3 Hz, 1H), 6.83 (dd,J=9.2, 4.0 Hz, 2H), 3.06 (d, J=1.2 Hz, 6H). ¹³C NMR (101 MHz, dmso) δ165.21, 154.76, 153.35, 149.88, 144.06, 143.10, 134.80, 130.84, 129.26,128.79, 126.68, 125.79, 125.65, 125.51, 124.74, 122.60, 122.31, 122.13,121.98, 116.94, 112.03.

Synthesis ofN-(2-amino-5-(thiophen-2-yl)phenyl)-4-((4-(dimethylamino)phenyl)diazenyl)benzamide(11a)

Compound 10f was prepared following the procedure A where4-dimethylamino-azobenzene-4′-carboxylic acid (0.110 g, 0.408 mmol) wastreated with tert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate (9a)(0.13 g, 0.449 mmol) to prepare the anilinic amine Boc protectedintermediate 10f which was purified by column chromatography using 25%EtOAc in hexane solvent system. Boc deprotection of 10f was carried outusing trifluoroacetic acid and dichloromethane mixture (30:70) to getdesired final compound 11a (0.096 g, 53% yield). ¹H NMR (400 MHz,DMSO-d6) δ 8.16-8.04 (m, 2H), 8.00 (d, J=2.3 Hz, 1H), 7.90-7.77 (m, 4H),7.43 (dd, J=5.1, 1.2 Hz, 1H), 7.39-7.28 (m, 2H), 7.08 (dd, J=5.1, 3.5Hz, 1H), 6.96 (d, J=8.3 Hz, 1H), 6.83 (dd, J=9.2, 4.0 Hz, 2H), 3.06 (d,J=1.2 Hz, 6H). ¹³C NMR (101 MHz, dmso) δ 165.33, 154.60, 153.31, 144.67,143.60, 143.08, 135.14, 129.49, 128.67, 125.61, 124.47, 123.71, 123.65,122.64, 121.92, 121.45, 116.78, 112.03.

Synthesis ofN-(4-amino-[1,1′-biphenyl]-3-yl)-4-((4-(dimethylamino)phenyl)diazenyl)benzamide(11b)

This compound was made using the same synthetic protocol used to makecompound 11a. 4-Dimethylamino-azobenzene-4′-carboxylic acid (0.18 g,0.67 mmol) acid and tert-butyl (3-amino-[1,1′-biphenyl]-4-yl)carbamate(9b) (0.256 g, 1.34 mmol) were used to make the intermediate compound10g and Boc deprotection of this compound gave the final compound 11b(0.134 g, 46% overall yield). ³H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H),8.15 (d, J=8.2 Hz, 2H), 7.90-7.79 (m, 4H), 7.59-7.50 (m, 3H), 7.42-7.29(m, 3H), 7.23 (d, J=7.3 Hz, 1H), 6.86 (t, J=9.3 Hz, 3H), 5.13 (s, 2H),3.07 (s, 6H). ¹³C NMR (101 MHz, dmso) δ 165.31, 154.58, 153.31, 143.36,143.08, 140.61, 135.25, 129.47, 129.24, 128.51, 126.45, 125.93, 125.60,125.32, 125.23, 123.91, 121.93, 116.93, 112.03.

Synthesis of 4-acetamido-N-(4-amino-[1,1′-biphenyl]-3-yl)benzamides (13)

To make the compound 13, the above described general acid-amine couplingreaction conditions (A) had been followed. 4-acetamidobenzoic acid(0.044 g, 0.25 mmol) was treated with tert-butyl(3-amino-[1,1′-biphenyl]-4-yl)carbamate (9b) (0.07 g, 0.25 mmol) to makecompound 12 which was the exposed to Boc deprotection conditions andpurified by column chromatography using 30% ethylacetate in hexane getfinal compound 13 (0.063 g, 74% overall yield). ¹H NMR (400 MHz,DMSO-d6) δ 10.19 (s, 1H), 9.62 (s, 1H), 7.95 (d, J=8.3 Hz, 2H), 7.69 (d,J=8.4 Hz, 2H), 7.52 (dd, J=15.1, 4.9 Hz, 3H), 7.41-7.17 (m, 4H), 6.85(d, J=8.3 Hz, 1H), 5.06 (s, 2H), 2.07 (s, 3H). ¹³C NMR (101 MHz, dmso) δ169.18, 165.32, 143.21, 142.58, 140.63, 129.23, 129.16, 128.56, 126.43,125.93, 125.19, 125.01, 124.17, 118.45, 116.95.

Example 3—Compound Activity

IC₅₀ Assay

The inhibitory effect on deacetylase activity for the compounds providedherein was determined using the conditions from a published optimizedbiochemical assay (see, e.g., Bradner, J. E. et al., Nature ChemicalBiology, 2010, 6, 238-243; and Bowers, A. et al., J Am Chem Soc 2008,130, 11219-11222). Compounds were added to white 96-well platescontaining 40 μL of HDAC assay buffer (50 mM HEPES, 100 mM KCl, 0.05%BSA, 0.001% Tween-20 at pH 7.4) by pin transfer. To the assay plate wasadded 40 μL of a stock solution of full-length HDAC protein at threetimes the desired final concentration. After addition the plate wascentrifuged to mix the solution while removing air bubbles. Our 470 nm(22.9V/0.2 A) LED 96-well excitation device (FIG. 4 ) was fitted atop ofthe assay plate inducing photoisomerization to the cis isomer of thecompounds while preincubating with the enzyme for 1 hour. After which 40μL of an HDAC buffer solution containing 3× trypsin and 3× the Kmconcentration of substrate was added. The plate was centrifuged andimmediately read on a Tecan Satire II plate reader (ex. 345 nm, em 440nm, Gain 60) representing the initial read for start of the assay. Afterwhich, reads were taken every 15 minutes to follow the fluorogenicrelease of 7-amino-4-methylcoumarin from the substrate resulting fromdeacetylase and trypsin enzymatic activity. In between reads the LEDexcitation device was reaffixed on top of the plate ensuring maximumcis-isomer population. In parallel to this, a second plate was preparedidentically and measured for activity but this plate was not excited byour device but rather was covered to prevent any ambient light fromshining on the plate. Replicate pairs of plates were performed.

Half-Life Experiment

To directly compare the residence time versus isoform relaxation timeBG47 and BG48 were incubated with HDAC1 in alternating columns of awhite 96-well plate. Columns 1 and 2 were exposed to 470 nm light(22.9V/0.2 A; 0.1 A per column) from LEDs using our excitation device 17hours before the assay for 1 hour, resulting in 16 hours of relaxationtime before the assay. Columns 3 and 4 were exposed to light 9 hoursbefore the assay for 1 hour providing an 8 hour relaxation period andcolumns 5 and 6 were exposed 5 hours before the assay providing a 4 hourrelaxation time point while columns 11 and 12 were used as controls withno pre-exposure. After the completion of the pre-exposure and relaxationtime points a stock solution of trypsin and substrate was added to theplate. The plate was centrifuged and read using a Tecan Safire II every2 minutes while keeping the plate inside the instrument, eliminatingexposure to ambient light.

To allow for high throughput profiling in 96-well microtiter plateformat a 12×8 LED array was developed to match the standardizedmicrotiter plate layout (FIG. 4 ). The LED arrays were connected to amicro controller that was programmed to control individual LEDs, LEDrows, or LED columns within the array. The controller allowed formodulation of LED intensity by controlling the on/off timing (PWM) or bydirectly controlling the supplied voltage. The temporal control was <10ms. The LEDs and microcontroller were powered optionally by a powersupply unit connected to a standard 110 V outlet or by a battery-pack,which allowed for use in a cell culture incubator. The LED array wasused as lid to illuminate plates from the top and as a stage toilluminate the plates from the bottom, as required by the experimentalsetup.

Compounds were profiled physically to determine absorbance spectra,solubility and kinetics of photo-isomerization kinetics and thermalrelaxation (where applicable). The compounds were then profiledbiochemically to determine the HDAC inhibitory profile as a function oflight exposure. Finally, selected compounds were profiled in cellularassays for the ability to induce hyperacetylation of histone proteins,which represents a biomarker readout for HDAC inhibition.

Compounds studied include those analogs with a push-pull azobenzenesystem (BG14, BG18, BG47, BG48 and BG49). These compounds absorbed inthe blue range of the visible spectrum, shown in FIG. 5 for BG14 andBG18. In contrast BG12 and BG19, which do not feature a push-pullsystem, maximally absorbs in the UV range (FIG. 5 ).

The compound panel was also profiled for inhibitory activity againstHDAC1-3 with and without exposure (including 1 hr pre-exposure) to 470nm light (FIGS. 6-8 ). Significantly increased activity was observed forBG14, BG18, BG47, BG48 and BG49 in samples exposed to light, while nodifferential activity was seen for the control compound CI-994.

The activation of the photoswitchable inhibitors depends on the lightenergy and the exposure time (as predicted by our model). FIG. 9compares the activity change of BG14 and the reference HDAC inhibitorsMS-275 and SAHA as a function of exposure time. Following pre-incubationof the HDAC enzyme and respective small molecule inhibitor, the enzymesubstrate was added and the assay plate was continuously exposed tolight (470 nm). Inhibitory activities were measured in 15 min intervals.Only compound BG14 shows increased activity with increased exposuretime.

The benzamide inhibitors exhibit long residence time once engaged withthe respective HDAC enzyme as shown for BG47 and BG48 (FIG. 10 ). Bothcompounds were incubated at 520 nM with HDAC1, exposed for 1 hour of 470nM light and stored in the dark for 4, 8 and 17 hours, respectively,followed by addition of enzyme substrate. The activity was monitored inreference to unexposed compound with continuous readout measured over 2hours, estimating a residence half-live of 12-24 hours.

The activity of the compounds in live cells was validated usingacetylation of histone H3 Lysine 9 as a biomarker for HDAC inhibition.As shown in FIG. 11 , using BG49 as a model compound, statisticallysignificant induced hyperacetylation of H3K9 relative to DMSO controlwas observed only following light exposure. The stronger induction bySAHA is the consequence of additional inhibition of HDAC3, which is nottargeted by BG49.

Example 4—Preparation of Compounds

Synthesis of Methyl 7-(4-(phenyldiazenyl)benzamido)heptanoate (15a)

Compound 15a was synthesized by acid amine coupling reaction using PyBOPas a coupling reagent. In a 50 mL round bottom flask, commerciallyavailable 4-(phenyldiazenyl)benzoic acid (0.10 g, 0.442 mmol) wassuspended in 20 mL of dry dichloromethane and methyl 7-aminoheptanoate(0.07 g, 0.442 mmol) was added into it. In the reaction vessel PyBOP(benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate)(0.28 g, 0.53 mmol) and DIPEA (N,N′-diisopropylethyldiamine) (0.085 mL,d=0.742 g/mL, 0.47 mmol) were added and the reaction mixture was stirredat room temperature in the dark for 2 hours. The reaction mixture wasdiluted with 200 mL dichloromethane and washed with 3×100 mL water. Theorganic layer was evaporated under vacuum and purified by columnchromatography using hexane:ethyl acetate solvent mixture to yielddesired product (92 mg, 57%).

Synthesis of Methyl7-(4-((4-(dimethylamino)phenyl)diazenyl)benzamido)heptanoate (15b)

Compound 15b was synthesized following the above mentioned procedurewhere 4-((4-(dimethylamino)phenyl)diazenyl)benzoic acid (0.12 g, 0.45mmol) and methyl 7-aminoheptanoate (0.071 g, 0.446 mmol) were used tothe desired product (0.10 g, 55%).

Synthesis ofN-(7-(hydroxyamino)-7-oxoheptyl)-4-(phenyldiazenyl)benzamide (16a)

Methyl ester 15a (0.092 g, 0.25 mmol) was suspended in a 4 mL of 1:1(v/v) mixture of methanol and 50% hydroxylamine (aq). To this suspensionwas added 2 N NaOH(aq) (1.0 mL). After 2 hours the mixture had becomehomogeneous. Then 2 N HCl was added, bringing the solution back to aneutral pH, upon which the product precipitated from solution.Filtration of the solid afforded the product 16a (0.071 g, 77%).

Synthesis of4-((4-(dimethylamino)phenyl)diazenyl)-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide(16b)

Intermediate compound 15b (0.10 g, 0.24 mmol) was used to make the finalcompound 16b (0.073 g, 72.8%) using the same procedure followed to make16a.

Synthesis of methyl 8-oxo-8-((4-(phenyldiazenyl)phenyl)amino)octanoate(18a)

4-(phenyldiazenyl)aniline hydrochloride 17a (0.15 g, 0.64 mmol) andtriethylamine (268 μL, 1.93 mmol) were dissolved in DCM (10 mL) andcooled to 0° C. To this solution was added methyl8-chloro-8-oxooctanoate (109 μL, 0.77 mmol). After 1 hour the mixturewas diluted with DCM (50 mL) and then washed with H₂O (50 mL), brine (50mL) and then dried with magnesium sulfate. Purification by columnchromatography (silica gel, DCM/EtOAc 50%) gave product 18a (0.199 g,84%).

Synthesis of N¹-hydroxy-N⁸-(4-(phenyldiazenyl)phenyl)octanediamide (19a)

Compound 19a was synthesized using the same procedure that was followedto make 16a where methyl8-oxo-8-((4-(phenyldiazenyl)phenyl)amino)octanoate (18a, 0.192 g, 0.522mmol) was used to get the final compound 19a (0.112 g, 58.2%).

Synthesis of Methyl8-((4-((4-(dimethylamino)phenyl)diazenyl)phenyl)amino)-8-oxooctanoate(18b)

Compound 18b was made following the amidation reaction that was followedto make 18a. 4-((4-aminophenyl)diazenyl)-N,N-dimethylaniline 17b (0.15g, 0.624 mmol) and triethylamine (174 μL, 1.24 mmol) were dissolved inDCM (10 mL) and cooled to 0° C. To this solution was added methyl8-chloro-8-oxooctanoate (133 μL, 0.93 mmol). After 1 hour the mixturewas diluted with DCM (50 mL) and then washed with H₂O (50 mL), brine (50mL) and then dried with magnesium sulfate. Purification by columnchromatography (silica gel, DCM/EtOAc 50%) gave product 18b (0.235 gm,92%).

Synthesis ofN¹-(4-((4-(dimethylamino)phenyl)diazenyl)phenyl)-N⁸-hydroxyoctanediamide(19b)

The final compound 19b was synthesized by using the same procedure thatdescribed to make 18a where 18b (0.227 g, 0.553 mmol) was used asstarting material to make the final desired compound (0.17 g, 74.5%).

Example 5—Preparation of Compounds

General Procedure: Amide Coupling of Azophenyl-Benzoic Acid and THPProtected Hydroxyl Amine (A) and Deprotection to Get the CorrespondingFinal Compounds (B):

Phenyl azobenzoic acid (1 eq) andO-(tetrahydro-2H-pyran-2-yl)hydroxylamine (1.2 eq) were suspended indichloromethane. PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate) and DIPEA (N,N′-diisopropylethyldiamine) (1.2 eqeach) were added into the reaction mixture. The reaction was continuedwith stirring at room temperature for overnight. Dichloromethane 4×volume was added to the reaction vessel to dilute and washed with water.All organic fractions were combined and evaporated under vacuum. Theconcentrate was purified by column chromatography withdichloromethane:methanol:triethyl amine (95:4:1) solvent system topurify the compounds. The intermediate synthesized compounds weresubjected to deprotection in a 50:50 mixture of dichloromethane (DCM)and trifluoroacetic acid (TFA). The deprotection reaction was carriedout by stirring the purified protected hydroxyl compounds in DCM/TFA (10mL for 100 mg compound) at room temperature for 30 minutes. The reactionmixture was evaporated under vacuum and sticky brown mass was suspendedin ethyl acetate, washed with saturated sodium bicarbonate solution.Evaporation of organic layer gave the sufficiently pure final compounds.

Synthesis of N-hydroxy-4-(phenyldiazenyl)benzamide (21a)

Compound 20a was synthesized following the general procedure (A)described above. In that procedure 4-(phenyldiazenyl)benzoic acid (0.10g, 0.442 mmol) was treated withO-(tetrahydro-2H-pyran-2-yl)hydroxylamine (0.062 g, 0.53 mmol) to getthe desired compound 20a (0.098 g, 68%) which was then exposed toTHP-deprotection reaction (B) described above to get the final compound21a (0.068 g, 93.6%).

Synthesis of 4-((4-(dimethylamino)phenyl)diazenyl)-N-hydroxybenzamide(21b)

The intermediate compound 20b was synthesized following the describedprocedure (A). 4-((4-(dimethylamino)phenyl)diazenyl)benzoic acid (0.15g, 0.557 mmol) was treated withO-(tetrahydro-2H-pyran-2-yl)hydroxylamine (0.065 g, 0.557 mmol) to getthe desired product 20b (0131 g, 64%) and deprotection of which gave thedesired final compound 21b (0.016 g, 16%).

Synthesis of 4-((4-(hydroxycarbamoyl)phenyl)diazenyl)benzoic Acid (21c)

Compound 20c was made using the general procedure (A) described above.There 4,4′-(diazene-1,2-diyl)dibenzoic acid (0.15 g, 0.555 mmol) andO-(tetrahydro-2H-pyran-2-yl)hydroxylamine (0.065 g, 0.555 mmol) wereused to get 20c (0.063 g, 31%) and that was exposed to the deprotectioncondition to get the final compound 21c (0.0305 g, 62.7%).

Synthesis of 3-((3-(hydroxycarbamoyl)phenyl)diazenyl)benzoic Acid (21d)

Compound 20d was made using the general procedure (A) described above.There 3,3′-(diazene-1,2-diyl)dibenzoic acid (0.15 g, 0.555 mmol) andO-(tetrahydro-2H-pyran-2-yl)hydroxylamine (0.065 g, 0.555 mmol) wereused to get 20d (0.077 g, 37.5%) and that was exposed to thedeprotection condition to get the final compound 21d (0.023 g, 38.7%).

Example 6—Preparation of(E)-N-(2-(2-((4-((4-((2-aminophenyl)carbamoyl)phenyl)diazenyl)phenyl)(methyl)amino)ethoxy)ethyl)-11-oxo-2,3,5,6,7,11-hexahydro-1H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxamide(SAS130)

To a solution of BG68 (0.0115 mmol, 1 eq.)

in DCM (2 mL), DIPEA (3 eq.) was added, followed by Coumarin 343 (1 eq.)and PYBOP (1.1 eq.). The reaction mixture was stirred for 30 minutes atroom temperature. After completion of the reaction, as monitored byLC/MS, the solvent was removed. The crude product was purified via flashchromatography (100% DCM→10:1 DCM: MeOH) yielding a dark orange solid(3.2 mg, 0.0045 mmol, 39%). ¹H NMR (400 MHz, Chloroform-d) δ 8.57 (s,1H), 8.0 (d, J=8.29 Hz, 2H), 7.89 (d, J=8.55 Hz, 2H), 7.86 (d, J=9.13Hz, 2H), 7.38 (d, J=7.95 Hz, 1H), 7.14-7.08 (m, 1H), 6.98 (s, 1H),6.90-6.94 (m, 2H), 6.78 (d, J=9.18 Hz, 2H), 3.90 (brs, 1H-NH), 3.70 (dd,J=4.37; 10.76 Hz, 4H), 3.63 (d, J=2.44 Hz, 4H), 3.34-3.29 (m, 4H), 3.16(s, 3H), 2.89 (t, J=6.42 Hz, 2H), 2.75 (t, J=6.14 Hz, 2H), 2.01-1.92 (m,4H). LC/MS: C₄₀H₄₁N₇O₅ Exact mass: 699.32; ES−[M]⁻ Found: 698.02;ES+[M]⁺ Found: 700.18.

Example 7—Inhibitor Characterization and Biochemical Profiling

Physical characterization of the prototype inhibitors in PBS confirmedthat all compounds strongly absorb light in the blue spectrum withmaximum absorbance around 470 nm.

Thermal relaxation half-lives of push-pull azobenzene analogs similar tothe presented compounds in aqueous solution at physiological pH have notbeen reported. Previous approaches to study fast cis-to-transisomerization are based on laser flash photolysis to measure thetransient absorbance change following excitation with a short laserpulse. Such experimental setups offer picosecond resolution, however,are very expensive and not commonly accessible. A readily adaptableinstrumentation setup was designed to measure relaxation kinetics withmicrosecond resolution. Using this approach the thermal relaxationhalf-lives of the studied HDAC ligands were found to range from 55-60μs, relative to 47 μs for 4-((4-(dimethylamino)phenyl)azo)benzoic acid(DABCYL), which is approximately 120× faster than previous measurementsfor DABCYL in chloroform (FIG. 13A). The estimate distance of diffusionof small molecules (diffusion coefficient D=10-6-10-7 cm²/s) at 10×T1/2(500 μs) in water is 0.1-0.3 μm, limiting diffusion of “activated”inhibitors well within subcellular dimensions.

To allow for high-throughput profiling of biochemical and cellularactivity a microprocessor-controlled (using the open-source Arduinoplatform) 12×8 LED-arrays that are compatible with 96-well microtiterplates was developed (As described above and further detailed in FIG.13B).

Using the LEDs array the light dependent HADC inhibitory activity of thereported compounds was accurately profiled. As shown in FIG. 13C-F,following light exposure all compounds strongly inhibited HDAC3enzymatic activity in a biochemical assay using an artificialacetyl-lysine tripeptide based on acH4K12, while no differentialactivity was observed for the reference HDAC inhibitor CI-994. Profilingof the inhibitor set against HDAC3 revealed that only BG14 showed potentlight-dependent inhibitory activity. In contrast, and as predicted for4-aryl substituted benzamide HDAC inhibitors, BG47 and BG48 did notinhibit HDAC3 in the presence and absence of light, which validates thatthe inhibitory activity is not the result non-specific inhibition.Furthermore, as shown for BG14 and HDAC3, the inhibitory activity isdirectly proportional to the average light exposure, demonstrating thatthe inhibitory potency can be directly controlled by light exposure.

Example 8—Inhibitor In Vitro Profiling

BG14, BG47 and BG48 were profiled in cell culture to assess cellularactivity using acetylation of histone H3 Lysine 9 (H3K9Ac) as apharmacodynamic marker for HDAC inhibition. As shown in FIG. 14 ,compound treatment of MCF7 cells in the presence of light stronglyinduced acetylation of H3K9 relative to the vehicle control. H3K9acetylation levels directly correlated with light exposure. Nosignificant histone acetylation above background was observed in theabsence of light. As expected, BG14, which also inhibits HDAC3, inducedthe strongest increase of H3K9 acetylation compared to theHDAC1/2-selective inhibitors BG47 and BG48.

Example 9—Expression Profiling

In order to gain more quantitative insights into the specific genesregulated by the presented inhibitors, and to provide further evidencethat the BG inhibitors elicit biological responses similar to theclosely related, but light-independent HDAC inhibitors a high-throughputgene expression analysis was performed. Expression profiling wasperformed in quadruplicate using the L1000™ platform (Genometry, Inc.).Specifically MCF7 cells were treated with various compounds and thetranscriptional changes were measured as a function of compoundconcentration, light exposure intensity, and treatment duration relativeto the reference HDAC inhibitors CI994 and “Merck60”.

Gene Set Enrichment Analysis (GSEA) comparing the expression patterns ofthe BG inhibitors with the reference inhibitors demonstrated a highcorrelation between the respective expression profiles under lighttreatment conditions, establishing strong support for similar biologicalactivity (FIG. 4B for BG14 and SI figures for BG47, BG48).

Interestingly, a lower but significant correlation was observed betweenphotochromic and reference HDAC inhibitors in the absence of light.Given the low affinity of the BG compounds for HDACs and the absence ofmeasurable increase of histone acetylation at the tested concentrationssuggests that transcriptional changes are possibly caused by off-targeteffects that are common to the tested inhibitors, which all share highchemical similarity.

Polypharmcology (DOI: 10.1021/jm400856t) of small molecules is wellrecognized and inhibition of unintended/unknown targets can contributeto the global biological response. Unintended inhibition of secondarytargets can complicate Chemical Genetics approaches using smallmolecules to dissect protein function. Commonly accepted approaches relyon comparative normalization to “inactive” analogs that establish abackground signature. In this context, photochromic ligands offer asignificant advantage as they allow one to compare active and inactivestates for a specific protein using the same chemical entity rather thancomparing two distinct molecules with sometimes significantly differentphysical and chemical properties.

Following this approach, subsets of transcripts that are onlydifferentially expressed in the presence of both light and inhibitorwere identified. The relative expression changes directly correlatedwith average light exposure intensity and inhibitor concentration (seeFIG. 15C,D). Importantly, light or inhibitor treatment alone does notinduce significant change in the respective transcript levels.

As shown in FIG. 15 , a L1000 platform was used to evaluate genesignatures in response to BG14 treatment under different light exposureconditions in MCF7 cell lines. In the absence of light, the vastmajority of landmark genes on the L1000 did not show any significantchanges in transcript levels. However, increasing amounts of lightresulted in significant changes of expression levels of specificlandmark genes, such as PAK1mRNA (FIG. 15A) and the cell division geneCDK2 (FIG. 15B), indicating that BG14-cis to BG14-trans transformationsuccessfully occurred in MCF7 cells and is a prerequisite for itsactivity. To verify the light-dependent nature of BG14's activity, thetop 100 up and top 100 downregulated transcripts (both landmark andinferred genes) were identified. Very strikingly, the relative heatmap(FIG. 15C) depicts very little change in transcript levels in absence oflight, irrespective of compound concentration, while with increasingamounts of light both a light-dose and concentration-dependent change inexpression levels was observed, thus suggesting that light-dependentcontrol of BG14 activity holds true for a large number of genes.

To assess BG14's nature as an HDAC inhibitor beyond its ability tomodulate HDAC and histone acetylation levels, Gene Set EnrichmentAnalysis (GSEA) was performed between BG14 (max concentration, maxamount of light) and the known HDAC inhibitor CI-994 (the sameconcentration) (FIG. 15D). Very remarkably, the Normalized EnrichmentScore (NES) of 3.25 indicates a very high degree of similarity betweenboth expression profiles. The identified 177 On-Target Gene List,(landmark genes regulated by light-dependent BG14_trans only), as wellas the 68 Off-Target list (landmark genes regulated in absence of lightby BG14_cis) were compared to the list of genes regulated by CI-994. 64%of the On-Target genes overlap with CI-994's gene list (FIG. 15E).Genemania-derived network and pathway analysis was performed on the‘Core Gene Set’ and multiple significant networks were identified. Allidentified significant features/networks are related to aspects of cellcycle and mitochondrial regulation and function. Furthermore, the genesin the cell cycle and mitochondria networks consist of the top 25downregulated genes were within the Core Gene set (FIG. 15F,G).

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A compound selected from the group consisting of:

or a pharmaceutically acceptable salt form thereof.
 2. A compoundselected from any one of the following compounds:

or a pharmaceutically acceptable salt thereof.
 3. A compound selectedfrom:

or a pharmaceutically acceptable salt thereof.